EA046169B1 - PLANT ORGANISM ENRICHED IN OLEIC ACID, HAVING GENETICALLY MODIFIED FAD2, AND METHOD FOR ITS OBTAINING - Google Patents

Description

Technical field of the invention

The present invention relates to the manipulation or modification of the FAD2 gene using the CRISPR-Cas system to increase the oleic acid content of a plant organism and, in particular, to a plant organism in which the oleic acid content is increased by modifying the FAD2 gene using the CRISPR-Cas system , capable of targeting the corresponding gene, to a composition intended to carry out manipulation, capable of manipulating the FAD2 gene, and to a method of using it.

BACKGROUND OF THE INVENTION

Soybean oil is the second most consumed edible oil in the world due to its richness in essential fatty acids and the high utilization of soybean meal as a by-product with an annual production of 45 million tons. Approximately 62% of the fatty acids contained in soybean oil are polyunsaturated fatty acids (PUFA), and 54% of the fatty acids are linoleic acid, and 8% of the fatty acids are linolenic acid. Because fatty acids have two or more double bonds, oxidation occurs easily and the oil quickly becomes rancid, making it difficult to store and distribute. (It does not store well and does not sell well.) Consequently, pre-treatment and refining methods are required to produce consistent quality soybean oil for use in processing or cooking. Most soybean oil producers maintain a certain level of quality by preventing rancidity by treating it with partial hydrogenation to add hydrogen to the unsaturated double bond, with oxidation easily accomplished during the production process.

However, partial hydrogenation has the disadvantage that it produces trans fatty acids, which pose a risk to the method of saturating the double bond of unsaturated fatty acids with hydrogen. Although in the natural state the formation of cis fatty acids is predominant by oxidation, and since the transform forms are thermodynamically stable, trans fatty acids, which are geometric isomers not existing in nature, are formed by hydrogenation or processing.

Due to controversy over the risks posed by trans fats, in 2015 the US FDA decided to remove this partially hydrogenated oil, which is widely used in processed foods, from its Generally Recognized as Safe (GRAS) list. Accordingly, US food manufacturers are seeking various edible oils that can replace and eliminate the use of partially hydrogenated oils by 2018, and the related industry is expected to spend US$6 billion to provide an alternative edible oil or new manufacturing process. In the US alone, based on FDA activity, demand for partially hydrogenated soybean oil is expected to decline by 9 million tons per year.

In addition, many countries such as Europe, Korea and Japan, as well as the United States, are well aware of the risks posed by trans fatty acids and are tending to encourage people to avoid consuming it whenever possible, with global regulations in the future regulations regarding partially hydrogenated oil will obviously be tightened. Therefore, there is a need to develop edible oil products that do not contain trans fatty acids that can replace soybean oil produced by partial hydrogenation.

Disclosure of the present invention

Technical tasks.

In order to solve the above-described objects, the present invention provides a plant containing a manipulated genome, wherein the manipulated genome comprises a manipulated FAD2 gene in which an insertion-deletion mutation in a sequence selected from SEQ ID NO: 1-30 is induced by a system CRISPR/Cas, wherein the sequence selected from SEQ ID NO: 1-30 comprises the PAM sequence 5'NGG-3', wherein N is independently A, T, C or G, wherein the manipulated FAD2 gene is capable of knocking out the FAD2 gene wild-type, thereby inhibiting the expression of the wild-type FAD2 gene in the plant, wherein the plant containing the artificially manipulated gene, compared to the wild-type plant, is characterized by at least one phenotype selected from the group consisting of:

(a) decreased FAD2 protein expression;

(b) reduced amount of RNA transcripts of the FAD2 gene;

(c) increased content of C8-24:D1 unsaturated fatty acid; and (d) decreased content of C8-24:D2 unsaturated fatty acid.

In a preferred embodiment, the C8-24:D1 unsaturated fatty acid is a C16-22:D1 unsaturated fatty acid.

- 1 046169

In yet another preferred embodiment, the C8-24:D1 unsaturated fatty acid is a C18:D1 unsaturated fatty acid.

In yet another preferred embodiment, the C8-24:D2 unsaturated fatty acid is a C16-22:D2 unsaturated fatty acid.

In yet another preferred embodiment, the C8-24:D2 unsaturated fatty acid is a C18:D2 unsaturated fatty acid.

In yet another preferred embodiment, the plant is soybean.

The present invention also provides a composition for producing a plant containing an artificially manipulated genome, which contains:

a guide nucleic acid capable of targeting the FAD2 gene, or a nucleic acid sequence encoding the same, and an editing protein or a nucleic acid sequence encoding the same, where the editing protein is a Cas9 protein derived from Streptococcus pyogenes, where the guide nucleic acid is an RNA sequence capable of binding a target region within the nucleic acid sequence of the FAD2 gene, wherein the target gene region contains at least one nucleotide sequence selected from SEQ ID NO: 1-30.

In a preferred embodiment, the composition for gene manipulation is formulated in an Agrobacterium-based vector system.

In yet another preferred embodiment, the composition for gene manipulation is formulated in a viral vector system.

In yet another preferred embodiment, the viral vector includes one or more viral vectors selected from mosaic virus, retrovirus, lentivirus, adenovirus, adeno-associated virus (AAV), vaccinia virus, poxvirus and herpes simplex virus.

The present invention also provides a method for producing a plant containing the artificially manipulated gene described above, comprising delivering the composition to a subject, where the subject is a plant cell, a seed, a part of a plant organism, or an entire plant organism, wherein the composition contains:

a guide nucleic acid capable of targeting the FAD2 gene, or a nucleic acid sequence encoding the same, and an editing protein or a nucleic acid sequence encoding the same, where the editing protein is a Cas9 protein derived from Streptococcus pyogenes, where the guide nucleic acid is an RNA sequence capable of binding a target region within the nucleic acid sequence of the FAD2 gene, wherein the target gene region contains at least one nucleotide sequence selected from SEQ ID NO: 1-30.

In a preferred embodiment, the composition is formulated in an Agrobacterium-based vector system.

In yet another preferred embodiment, the composition is formulated in a viral vector system.

In yet another preferred embodiment, the viral vector includes one or more viral vectors selected from mosaic virus, retrovirus, lentivirus, adenovirus, adeno-associated virus (AAV), vaccinia virus, poxvirus and herpes simplex virus.

The present invention relates to an artificially manipulated unsaturated fatty acid control system that has the effect of increasing the content of a specific unsaturated fatty acid. More particularly, the present invention relates to an artificially manipulated unsaturated fatty acid biosynthesis-associated factor and an unsaturated fatty acid control system that artificially modifies the content of a specific unsaturated fatty acid.

The present invention is directed to providing a plant organism with an increased content of a specific unsaturated fatty acid provided by an artificially manipulated factor associated with the biosynthesis of unsaturated fatty acids.

The present invention is directed to providing a plant organism with a reduced content of a specific unsaturated fatty acid provided by an artificially manipulated unsaturated fatty acid biosynthesis associated factor.

In an illustrative embodiment of the present invention, the present invention provides an artificially manipulated unsaturated fatty acid biosynthesis-associated factor.

In an illustrative embodiment of the present invention, the present invention provides a manipulable unsaturated fatty acid control system.

In an illustrative embodiment of the present invention, in the present invention

- 2 046169 presents a factor associated with the biosynthesis of unsaturated fatty acids, subjected to artificial manipulation, and the product of its expression.

In an illustrative embodiment of the present invention, the present invention provides a composition for performing gene manipulation for manipulating an unsaturated fatty acid biosynthesis-associated factor, and a method for using it.

In an illustrative embodiment of the present invention, the present invention provides a method for controlling the biosynthesis of an unsaturated fatty acid.

In an illustrative embodiment of the present invention, the present invention provides a method for controlling the type of unsaturated fatty acid and its content.

In an illustrative embodiment of the present invention, the present invention provides an unsaturated fatty acid control composition for the purpose of controlling unsaturated fatty acid biosynthesis and/or unsaturated fatty acid content and various uses thereof.

In an illustrative embodiment of the present invention, the present invention provides an artificially manipulated unsaturated fatty acid biosynthesis-associated factor such as FAD2, FAD3, FAD4, FAD6, FAD7 or FAD8, and/or an expression product thereof.

In an illustrative embodiment of the present invention, the present invention provides a composition for gene manipulation to artificially manipulate an unsaturated fatty acid biosynthesis-associated factor such as FAD2, FAD3, FAD4, FAD6, FAD7 or FAD8.

In an illustrative embodiment of the present invention, the present invention provides an artificially manipulated unsaturated fatty acid biosynthesis-associated factor such as FAD2, FAD3, FAD4, FAD6, FAD7 or FAD8, and/or various uses of a gene manipulation composition for the purpose of artificial manipulation.

In an illustrative embodiment of the present invention, the present invention provides a plant organism with increased or decreased content of a specific unsaturated fatty acid and a processed product thereof.

Solutions to technical problems.

To address these objectives, the present invention provides a system capable of artificially controlling unsaturated fatty acid biosynthesis and/or fatty acid content, which includes an artificially manipulated unsaturated fatty acid biosynthesis-associated factor and/or a composition capable of artificially manipulating the unsaturated fatty acid. a factor associated with the biosynthesis of unsaturated fatty acids in order to control the content of a specific unsaturated fatty acid.

In one illustrative embodiment of the present invention, a plant organism is provided with an increased content of a specific unsaturated fatty acid provided by an artificially manipulated unsaturated fatty acid biosynthesis associated factor.

In another illustrative embodiment, the present invention provides a specific unsaturated fatty acid obtained from a plant organism using an artificially manipulated unsaturated fatty acid biosynthesis associated factor.

The term specific unsaturated fatty acid as used herein refers to one or more unsaturated fatty acids selected from various types of known unsaturated fatty acids, which may be one or more unsaturated fatty acids selected from a classification system represented by the number of carbon atoms ( C) and the number of double bonds (D) that are included in unsaturated fatty acids, among the different types of unsaturated fatty acids. The term CN:DM unsaturated fatty acid as used herein refers to an unsaturated fatty acid consisting of N number of carbon atoms (C) and including M number of double bonds (D). Here, N may be an integer from 4 to 36, and M may be an integer from 1 to 35.

The specific unsaturated fatty acid may be a C8-24:D1 unsaturated fatty acid.

The specific unsaturated fatty acid may be a C16-22:D1 unsaturated fatty acid.

The specific unsaturated fatty acid may be a C18:D1 unsaturated fatty acid.

The specific unsaturated fatty acid may be oleic acid, elaidic acid or vaccenic acid.

In addition, the specific unsaturated fatty acid may be C8-24:D2 not

- 3 046169 saturated fatty acid.

The specific unsaturated fatty acid may be a C16-22:D2 unsaturated fatty acid.

The specific unsaturated fatty acid may be a C18:D2 unsaturated fatty acid.

The specific unsaturated fatty acid may be linoleic acid or linoleaidic acid.

In one illustrative embodiment of the present invention, the present invention provides an artificially manipulated unsaturated fatty acid biosynthesis-associated factor.

The term unsaturated fatty acid biosynthesis-associated factor as used herein refers to all factors directly involved in or indirectly affecting the biosynthesis of an unsaturated fatty acid. In this case, the factor may be DNA, RNA, gene, peptide, polypeptide or protein. The factor includes various materials capable of controlling the biosynthesis of an unsaturated fatty acid that are unnatural, that is, they are subject to artificial manipulation. For example, a factor may be a genetically manipulated or modified gene or protein that is expressed in a plant.

Unsaturated fatty acid biosynthesis-associated factor can increase the specific unsaturated fatty acid content of a plant.

A factor associated with the biosynthesis of unsaturated fatty acids can reduce the content of a specific unsaturated fatty acid contained in a plant.

Unsaturated fatty acid biosynthesis-associated factor may influence the direct/indirect mechanism to control the specific unsaturated fatty acid content of the plant.

In one illustrative embodiment of the present invention, the unsaturated fatty acid biosynthesis-associated factor may be, for example, a manipulated FAD2 gene, FAD3 gene, FAD4 gene, FAD6 gene, FAD7 gene, or FAD8 gene, preferably a FAD2 gene or a FAD3 gene.

In one illustrative embodiment of the present invention, the unsaturated fatty acid biosynthesis-associated factor may comprise two or more manipulated genes. For example, two or more genes selected from the group consisting of a FAD2 gene, a FAD3 gene, a FAD4 gene, a FAD6 gene, a FAD7 gene, and a FAD8 gene may be artificially manipulated.

Therefore, an illustrative embodiment of the present invention provides one or more artificially manipulated unsaturated fatty acid biosynthesis-associated factors selected from the group consisting of the FAD2 gene, the FAD3 gene, the FAD4 gene, the FAD6 gene, the FAD7 gene, and the FAD8 gene, which have been manipulated modifications in the nucleic acid sequence.

Modification in a nucleic acid sequence can be carried out without limitation by artificial manipulation using a guide nucleic acid editing protein complex.

The term guide nucleic acid-editing protein complex refers to a complex formed through the interaction between a guide nucleic acid and an editing protein, wherein the nucleic acid-protein complex includes a guide nucleic acid and an editing protein.

The guide nucleic acid-editing protein complex may serve to modify the subject. The subject may be a target nucleic acid, gene, chromosome or protein.

For example, the gene may be an unsaturated fatty acid biosynthesis-associated factor artificially manipulated by a guide nucleic acid-editing protein complex, wherein the artificially manipulated unsaturated fatty acid biosynthesis-associated factor includes one or more nucleic acid modifications that are at least one of deletions or insertions of one or more nucleotides, substitutions with one or more nucleotides other than those of a wild-type gene, and insertions of one or more foreign nucleotides, within a protospacer adjacent motif (PAM) sequence within the nucleic acid sequence that constitutes the biosynthesis-associated unsaturated fatty acid factor, or within a contiguous stretch of base sequence of 1 bp in length. up to 50 bp adjacent to its 5'-end and/or 3'-end, or chemical modification of one or more nucleotides in the nucleic acid sequence of which the unsaturated fatty acid biosynthesis-associated factor component is composed.

Modification of nucleic acids can be carried out in the promoter region of the gene.

Modification of nucleic acids can be carried out in the exonic region of the gene. In one illustration

- 4 046169 in a stratified embodiment, 50% of the modifications can be carried out at a site in the 3'-5' direction relative to the coding regions of the gene.

Modification of nucleic acids can be carried out in the intronic region of the gene. Modification of nucleic acids can occur in the enhancer region of a gene.

The PAM sequence may be, for example, one or more of the following sequences (described in the 5'-3' direction):

NGG (N represents A, T, C or G);

NNNNRYAC (N is each independently A, T, C or G, R is A or G, and Y is C or T);

NNAGAAW (N is each independently A, T, C or G, and W is A or T);

NNNNGATT (N is each independently A, T, C or G);

NNGRR(T) (N is each independently A, T, C or G, and R is A or G) and

TTN (N represents A, T, C or G).

The editing protein can be obtained from

Streptococcus pyogenes, Streptococcus thermophilus, Streptococcus sp., Staphylococcus aureus, Nocardiopsis dassonvillei, Streptomyces pristinaespiralis, Streptomyces viridochromogenes, Streptomyces viridochromogenes, Streptosporangium roseum, Streptosporangium roseum, Alicyclobacillus acidocaldarius, Bacil lus pseudomycoides, Bacillus selenitireducens, Exiguobacterium sibiricum, Lactobacillus delbrueckii, Lactobacillus salivarius, Microscilla marina, Burkholderiales bacterium, Polaromonas naphthalenivorans, Polaromonas sp., Crocosphaera watsonii, Cyanothece sp., Microcystis aeruginosa, Synechococcus sp., Acetohalobium arabaticum, Ammonifex degensii, Caldicelulosiruptor bescii, Candidatus Desulforudis, Clostridium botulinum, Clostridium diff icile, Finegoldia magna, Natranaerobius thermophilus, Pelotomaculum thermopropionicum, Acidithiobacillus caldus, Acidithiobacillus ferrooxidans, Allochromatium vino sum, Marinobacter sp., Nitrosococcus halophilus, Nitrosococcus watsonii, Pseudoalteromonas haloplanktis, Ktedonobacter racemifer, Methanohalobium evestigatum, Anabaena variabilis, Nodularia spumig ena, Nostoc sp., Arthrospira maxima, Arthrospira platensis, Arthrospira sp ., Lyngbya sp., Microcoleus chthonoplastes, Oscillatoria sp., Petrotoga mobilis, Thermosipho africanus or Acaryochloris marina.

In one illustrative embodiment, the editing protein may be one or more selected from the group consisting of a Cas9 protein derived from Streptococcus pyogenes, a Cas9 protein derived from Campylobacter jejuni, a Cas9 protein derived from Streptococcus thermophilus, a Cas9 protein derived from Staphylococcus aureus, the Neisseria meningitidis-derived Cas9 protein, and the Cpf1 protein. By way of example, the editing protein may be a Cas9 protein derived from Streptococcus pyogenes or a Cas9 protein derived from Campylobacter jejuni.

Additionally, in another embodiment, the present invention provides a guide nucleic acid that is capable of forming a complementary linkage to the target sequences of SEQ ID NO: 1-30, for example, SEQ ID NO: 7 or 30.

The guide nucleic acid may form a complementary bond with a portion of the nucleic acid sequences of the FAD2 gene. She can create 0-5, 0-4, 0-3 or 0-2 mismatches. As a preferred example, the guide nucleic acid may be nucleotides forming a complementary bond to one or more of the target sequences of SEQ ID NO: 1-30, for example, SEQ ID NO: 7 or 30, respectively.

The guide nucleic acid may include, but is not limited to, nucleotides of 18-25 bp, 1824 bp, 18-23 bp, 19-23 bp. or 20-23 bp

Furthermore, the present invention provides a gene manipulation composition that can be used to artificially manipulate an unsaturated fatty acid biosynthesis-associated factor for a specific purpose.

A composition intended for gene manipulation may include a complex of a guide nucleic acid - an editing protein or a nucleic acid sequence encoding it

- 5 046169 acid.

A composition intended for gene manipulation may contain:

(a) a guide nucleic acid capable of forming a complementary linkage to each of the target sequences of one or more genes selected from the group consisting of the FAD2 gene, the FAD3 gene, the FAD4 gene, the FAD6 gene, the FAD7 gene and the FAD8 gene, respectively, or a sequence a nucleic acid encoding a guide nucleic acid;

(b) an editing protein comprising one or more proteins selected from the group consisting of a Cas9 protein derived from Streptococcus pyogenes, a Cas9 protein derived from Campylobacter jejuni, a Cas9 protein derived from Streptococcus thermophilus, a Cas9 protein derived from Staphylococcus aureus, Neisseria meningitidis-derived Cas9 protein and Cpf1 protein, respectively, or a nucleic acid sequence encoding the same.

In one illustrative embodiment, the guide nucleic acid may be a nucleic acid sequence that forms a complementary linkage to one or more target sequences of SEQ ID NO: 1-30, respectively.

For example, the targeting nucleic acid may be a nucleic acid sequence that forms a complementary linkage to the target sequence of SEQ ID NO: 7 or 30.

In one illustrative embodiment, the composition intended for gene manipulation may be a viral vector system.

The viral vector may be an Agrobacterium-based vector system using Agrobacterium.

In one illustrative embodiment, the composition intended for gene manipulation may be a viral vector system.

The viral vector may include one or more viral vectors selected from the group consisting of mosaic virus, retrovirus, lentivirus, adenovirus, adeno-associated virus (AAV), vaccinia virus, poxvirus, and herpes simplex virus.

In one illustrative embodiment, the present invention provides a method of artificially manipulating a cell, which involves introducing into the cells (a) a targeting nucleic acid capable of forming a complementary bond to the target sequences of one or more genes selected from the group consisting of the FAD2 gene, the FAD3 gene, the FAD4 gene, the FAD6 gene, the FAD7 gene and the FAD8 gene, respectively, or a nucleic acid sequence encoding the same; and (b) an editing protein comprising one or more proteins selected from the group consisting of a Cas9 protein derived from Streptococcus pyogenes, a Cas9 protein derived from Campylobacter jejuni, a Cas9 protein derived from Streptococcus thermophilus, a Cas9 protein derived from Staphylococcus aureus , the Neisseria meningitidis-derived Cas9 protein and the Cpf1 protein, respectively, or a nucleic acid sequence encoding the same.

The guide nucleic acid and editing protein may be present in one or more vectors as a nucleic acid sequence or may be present in a complex formed by binding of the guide nucleic acid to the editing protein.

Administration may be performed in vivo or ex vivo relative to the plant.

Administration can be accomplished by one or more methods selected from gene gun bombardment, electroporation, liposomes, plasmids, Agrobacterium vector system, viral vectors, nanoparticles, and protein translocation domain (PTD) fusion method.

The viral vector may include one or more viral vectors selected from the group consisting of mosaic virus, retrovirus, lentivirus, adenovirus, adeno-associated virus (AAV), vaccinia virus, poxvirus, and herpes simplex virus.

In addition, the present invention provides a composition for controlling unsaturated fatty acid for the purpose of controlling the biosynthesis of unsaturated fatty acid and/or the content of unsaturated fatty acid in a plant.

The unsaturated fatty acid control composition may include a gene manipulation composition that can be used to artificially manipulate an unsaturated fatty acid biosynthesis-associated factor.

The composition of the gene manipulation composition is the same as described above.

In an illustrative embodiment, the present invention relates to a processed product using a plant organism with increased or decreased content of a specific unsaturated fatty acid.

A plant organism may contain a factor associated with the biosynthesis of unsaturated fatty acids that has been subjected to artificial manipulation.

The processed product may be a food product that can be consumed by humans and/or animals.

In an illustrative embodiment, the present invention provides a gene kit

- 6 046169 manipulation to control the content of a specific unsaturated fatty acid.

The kit may include a gene manipulation composition that can be used in the artificial manipulation of an unsaturated fatty acid biosynthesis-associated factor.

The gene of interest can be artificially manipulated using such a kit.

Positive effects.

A plant organism with an increased content of a specific unsaturated fatty acid that is beneficial to human health, or with a reduced content of a specific unsaturated fatty acid that is harmful to human health, and/or a processed product using it can be obtained using the associated biosynthesis of unsaturated fatty acids factor subjected to artificial manipulation, and a system for controlling the unsaturated fatty acid, which is thereby artificially modified.

For example, one or more genes selected from a FAD2 gene, a FAD3 gene, a FAD4 gene, a FAD6 gene, a FAD7 gene, and a FAD8 gene may be used.

Brief description of graphic materials

In fig. Figure 1 shows a schematic diagram of the CRISPR-Cas9, pPZP-FAD2-7 and pPZPFAD2-30 vectors designed to modify the soybean FAD2 gene.

In fig. Figure 2 shows the growth processes of transgenic soybean plant organisms obtained by knocking out the FAD2 gene using pPZP-FAD2-7(a) and pPZP-FAD2-30(b).

In fig. Figure 3 shows T ₀ transformants pPZP-FAD2-7 and pPZP-FAD2-30.

In fig. Figure 4 shows the results of PNR to confirm gene insertion in T ₀ transformants pPZPFAD2-7 and pPZP-FAD2-30. In this case, NT represents Glycine max L. Kwangan (wild type), and No. 1, No. 2, No. 3, No. 5, No. 8, No. 9, No. 19 and No. 21 are T ₀ transformants.

In fig. Figure 5 shows the oleic acid contents of T1 seeds of pPZP-FAD2-7 and pPZP-FAD2-30.

In fig. Figure 6 shows the frequency of insertion-deletion mutations of the FAD2 gene, which is targeted using CRISPR-Cas9.

In fig. 7 shows the results of sequencing of the FAD2 gene of soybean transformed using CRISPR-Cas9 (targeting sequence including PAM, shown underlined).

In fig. Figure 8 shows the results of screening for the target site and the frequency of insertion-deletion mutation of the FAD2 gene manipulated in T1 transformants, shows the results of screening for the target site and the frequency of insertion-deletion mutation of the FAD2 gene in (a) chromosome No. 10 (chr10) and (b) chromosome number 20 (chr20).

In fig. 9 shows the results of sequencing the target site of the FAD2 gene manipulated in T1 transformants, shows the results of sequencing the target site of the FAD2 gene in (a) chromosome No. 10 (chr10) and (b) chromosome No. 20 (chr20).

In fig. 10 shows T1 transformants pPZP-FAD2-7 and pPZP-FAD2-30.

In fig. 11 shows the results of a gene deletion assay from T1 transformants pPZP-FAD2-7 and pPZPFAD2-30 using PNR.

Embodiments of the Invention

Unless otherwise specified, all technical and scientific terms used herein have the same meanings as commonly known to those skilled in the art to which this invention relates. Although methods and materials similar or similar to those disclosed herein may be used, suitable methods and materials will be disclosed below in the implementation or experimentation of the present invention. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. Moreover, the materials, methods, and embodiments are illustrative only and are not intended to be limiting.

One aspect of the present invention relates to a transgenic plant organism with increased levels of C8-24:D1 unsaturated fatty acid.

In particular, the present invention relates to a transgenic plant organism with an increased content of a specific unsaturated fatty acid provided by an artificially manipulated unsaturated fatty acid biosynthesis-associated factor, the present invention comprising an artificially altered unsaturated fatty acid biosynthesis-associated factor, a composition , intended for artificial manipulation with it, the method of its preparation and the plant organism containing it.

Another aspect of the present invention relates to a transgenic plant organism with reduced levels of C8-24:D2 unsaturated fatty acid.

In particular, the present invention relates to a transgenic plant organism with reduced content of a specific unsaturated fatty acid provided by an artificially manipulated unsaturated fatty acid biosynthesis-associated factor, wherein the present invention includes an unsaturated fatty acid biosynthesis-associated factor

- 7 046169 acid factor, the function of which is artificially modified, a composition intended for artificial manipulation with it, a method for its preparation and a plant organism containing it.

Unsaturated fatty acid.

One aspect of the present invention relates to a system for modifying fatty acid content.

In one example, a system may be provided for modifying the content of a specific saturated fatty acid in a plant organism.

In another example, a system may be provided for modifying the content of a specific unsaturated fatty acid in a plant organism.

The term fatty acid as used herein refers to a carboxylic acid having an aliphatic chain, with most naturally occurring fatty acids having an even number of carbon atoms, ranging from about 4 to 36, forming a carbon chain. Fatty acids are mainly classified into saturated fatty acids and unsaturated fatty acids based on the type of carbon bond.

The term saturated fatty acid as used herein refers to fatty acids formed with a single bond.

Fatty acids include propionic acid, butyric acid, valeric acid, caproic acid, enanthic acid, caprylic acid, pelargonic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, arachidonic acid, behenic acid, lignoceric acid, cerotic acid and so on.

The term unsaturated fatty acids as used herein refers to fatty acids with one or more carbon-carbon double bonds. Unsaturated fatty acids include all cis-unsaturated fatty acids and trans-unsaturated fatty acids. Cis-unsaturated fatty acids refer to unsaturated fatty acids in which the two hydrogen atoms, respectively bonding to the two carbon atoms involved in the double bond, are structurally located in the same direction. On the other hand, trans-unsaturated fatty acids refer to unsaturated fatty acids in which the two hydrogen atoms respectively bonding to the two carbon atoms involved in the double bond are structurally located in different directions.

Unsaturated fatty acids can be classified into omega 3, 6, 7 and 9 according to the position of the carbon involved in the double bond.

Unsaturated fatty acids include omega-3 (ω-3) fatty acids.

As used here, the term omega-3 (ω-3) fatty acid refers to an unsaturated fatty acid in which the double bond begins at the third carbon at the end of the carbon chain, and includes alpha-linolenic acid (ALA), eicosapentaenoic acid (EPA), and docosahexaenoic acid. acid (DHA).

Unsaturated fatty acids include omega-6 (ω-6) fatty acids.

In this case, the term omega-6 (ω-6) fatty acids refers to unsaturated fatty acids in which the double bond begins at the sixth carbon at the end of the carbon chain, and includes linoleic acid (LA), gamma-linolenic acid (GLA) , dihomo-gamma-linolenic acid (DGLA) and arachidonic acid (AA).

Unsaturated fatty acids include omega-7 (ω-7) fatty acids.

As used herein, the term omega-7 (ω-7) fatty acids refers to unsaturated fatty acids in which the double bond begins at the seventh carbon at the end of the carbon chain and includes paulinic acid, palmitoleic acid or vaccenic acid.

Unsaturated fatty acids include omega-9 (ω-9) fatty acids.

As used herein, the term omega-9 (ω-9) fatty acids refers to unsaturated fatty acids in which the double bond begins at the ninth carbon at the end of the carbon chain, and includes oleic acid, elaidic acid, eicosenoic acid, erucic acid and nervonic acid.

In one illustrative embodiment, the unsaturated fatty acid may be an omega-6 (ω-6) fatty acid.

In another illustrative embodiment, the unsaturated fatty acid may be an omega-9 (ω-9) fatty acid.

In addition, an unsaturated fatty acid can be classified as a CN:DN unsaturated fatty acid based on the number of carbon atoms and the number of double bonds.

The term CN:DM unsaturated fatty acid refers to an unsaturated fatty acid consisting of N number of carbon atoms (C) and including M number of double bonds (D). Here, N may be an integer from 4 to 36, and M may be an integer from 1 to 35.

For example, an unsaturated fatty acid consisting of 18 carbon atoms and including 2 double

- 8 046169 nal bonds, can be classified as a C18:D2 unsaturated fatty acid.

In one illustrative embodiment, the unsaturated fatty acid includes a CN:D1 unsaturated fatty acid.

In this case, N can be an integer from 4 to 36.

Preferably, the CN:D1 unsaturated fatty acid may be a C8:D1 unsaturated fatty acid, a C10:D1 unsaturated fatty acid, a C12:D1 unsaturated fatty acid, a C14:D1 unsaturated fatty acid, a C16:D1 unsaturated fatty acid, a C18:D1 unsaturated fatty acid. acid, C20:D1 unsaturated fatty acid, C22:D1 unsaturated fatty acid, or C24:D1 unsaturated fatty acid.

In another illustrative embodiment, the unsaturated fatty acid includes a CN:D2 unsaturated fatty acid.

In this case, N can be an integer from 4 to 36.

Preferably, the CN:D2 unsaturated fatty acid may be a C8:D2 unsaturated fatty acid, a C10:D2 unsaturated fatty acid, a C12:D2 unsaturated fatty acid, a C14:D2 unsaturated fatty acid, a C16:D2 unsaturated fatty acid, a C18:D2 unsaturated fatty acid. acid, C20:D2 unsaturated fatty acid, C22:D2 unsaturated fatty acid or C24:D2 unsaturated fatty acid.

In yet another illustrative embodiment, the unsaturated fatty acid includes a CN:D3 unsaturated fatty acid.

In this case, N can be an integer from 4 to 36.

Preferably, the CN:D3 unsaturated fatty acid may be a C8:D3 unsaturated fatty acid, a C10:D3 unsaturated fatty acid, a C12:D3 unsaturated fatty acid, a C14:D3 unsaturated fatty acid, a C16:D3 unsaturated fatty acid, a C18:D3 unsaturated fatty acid. acid, C20:D3 unsaturated fatty acid, C22:D3 unsaturated fatty acid or C24:D3 unsaturated fatty acid.

In yet another illustrative embodiment, the unsaturated fatty acid includes a CN:D4 unsaturated fatty acid.

In this case, N can be an integer from 4 to 36.

Preferably, the CN:D4 unsaturated fatty acid may be a C8:D4 unsaturated fatty acid, a C10:D4 unsaturated fatty acid, a C12:D4 unsaturated fatty acid, a C14:D4 unsaturated fatty acid, a C16:D4 unsaturated fatty acid, a C18:D4 unsaturated fatty acid. acid, C20:D4 unsaturated fatty acid, C22:D4 unsaturated fatty acid or C24:D4 unsaturated fatty acid.

In yet another illustrative embodiment, the unsaturated fatty acid includes a CN:D5 unsaturated fatty acid.

In this case, N can be an integer from 4 to 36.

Preferably, the CN:D5 unsaturated fatty acid may be a C8:D5 unsaturated fatty acid, a C10:D5 unsaturated fatty acid, a C12:D5 unsaturated fatty acid, a C14:D5 unsaturated fatty acid, a C16:D5 unsaturated fatty acid, a C18:D5 unsaturated fatty acid. acid, C20:D5 unsaturated fatty acid, C22:D5 unsaturated fatty acid or C24:D5 unsaturated fatty acid.

In yet another illustrative embodiment, the unsaturated fatty acid includes a CN:D6 unsaturated fatty acid.

In this case, N can be an integer from 4 to 36.

Preferably, the CN:D6 unsaturated fatty acid may be a C8:D6 unsaturated fatty acid, a C10:D6 unsaturated fatty acid, a C12:D6 unsaturated fatty acid, a C14:D6 unsaturated fatty acid, a C16:D6 unsaturated fatty acid, a C18:D6 unsaturated fatty acid acid, C20:D6 unsaturated fatty acid, C22:D6 unsaturated fatty acid or C24:D6 unsaturated fatty acid.

In yet another illustrative embodiment, the unsaturated fatty acid includes a CN:DK unsaturated fatty acid.

Here, N may be an integer from 4 to 36, and K may be an integer from 7 to 35.

In one illustrative embodiment, the unsaturated fatty acid includes a C824:D1 unsaturated fatty acid.

Preferably, the unsaturated fatty acid may be selected from the group consisting of a C16:D1 unsaturated fatty acid, a C18:D1 unsaturated fatty acid, a C20:D1 unsaturated fatty acid and a C22:D1 unsaturated fatty acid.

Most preferably, the unsaturated fatty acid may be a C18:D1 unsaturated fatty acid or a C20:D1 unsaturated fatty acid.

In another illustrative embodiment, the unsaturated fatty acid includes a C824:D2 unsaturated fatty acid.

- 9 046169

Preferably, the unsaturated fatty acid may be selected from the group consisting of a C16:D2 unsaturated fatty acid, a C18:D2 unsaturated fatty acid, a C20:D2 unsaturated fatty acid and a C22:D2 unsaturated fatty acid.

Most preferably, the unsaturated fatty acid may be a C18:D2 unsaturated fatty acid or a C20:D2 unsaturated fatty acid.

Factor associated with the biosynthesis of unsaturated fatty acids.

Another aspect of the present invention relates to an unsaturated fatty acid biosynthesis-associated factor that has been artificially manipulated or modified.

The term unsaturated fatty acid biosynthesis-associated factor as used herein refers to all factors directly involved in or indirectly affecting the biosynthesis of an unsaturated fatty acid. In this case, the factor may be DNA, RNA, gene, peptide, polypeptide or protein.

In an illustrative embodiment, the unsaturated fatty acid biosynthesis-associated factor includes various materials capable of controlling unsaturated fatty acid biosynthesis that are non-natural, that is, artificially manipulated. For example, the unsaturated fatty acid biosynthesis-associated factor may be a genetically manipulated or modified gene or protein that is expressed in the plant.

The term artificially manipulated means an artificially modified condition, that is, a condition that does not exist in nature.

The term genetically manipulated means that genetic modification is artificially carried out in the plant materials referred to in the present invention, which may be, for example, genes and gene products (polypeptides, proteins, etc.) whose genomes have been artificially modified for a specific purpose.

As a preferred example, the present invention provides an unsaturated fatty acid biosynthesis-associated factor that is genetically manipulated or modified for a specific purpose.

Genes or proteins that have the following functions may have many types of functions, rather than just one type of function associated with the biosynthesis of unsaturated fatty acids. In addition, two or more functions and two or more factors associated with the biosynthesis of unsaturated fatty acids may be presented if necessary.

An unsaturated fatty acid biosynthesis-associated factor can initiate the production of an unsaturated fatty acid by forming one or more double bonds in a saturated fatty acid.

An unsaturated fatty acid biosynthesis-associated factor can initiate the formation of new one or more double bonds in an unsaturated fatty acid.

An unsaturated fatty acid biosynthesis-associated factor may change the position of one or more double bonds included in an unsaturated fatty acid.

An unsaturated fatty acid biosynthesis-associated factor may initiate the removal of one or more double bonds of an unsaturated fatty acid with two or more double bonds.

An unsaturated fatty acid biosynthesis-associated factor can initiate the conversion of a cis-unsaturated fatty acid to a trans-unsaturated fatty acid.

An unsaturated fatty acid biosynthesis-associated factor can initiate the conversion of a trans-unsaturated fatty acid to a cis-unsaturated fatty acid.

Unsaturated fatty acid biosynthesis-associated factor may control the amount of unsaturated fatty acid contained in the plant.

Unsaturated fatty acid biosynthesis-associated factor can increase the specific unsaturated fatty acid content of a plant.

A factor associated with the biosynthesis of unsaturated fatty acids can reduce the content of a specific unsaturated fatty acid contained in a plant.

In an illustrative embodiment, the unsaturated fatty acid biosynthesis-associated factor may be linked to a plant unsaturated fatty acid biosynthesis-associated factor.

Preferably, the unsaturated fatty acid biosynthesis-associated factor may be a FAD gene or a FAD protein.

Most preferably, the unsaturated fatty acid biosynthesis-associated factor may be one or more selected from the group consisting of FAD2, FAD3, FAD6, FAD7 and FAD8.

In any illustrative embodiment, the unsaturated fatty acid biosynthesis-associated factor may be FAD2.

The FAD2 (omega-6 fatty acid desaturase) gene refers to a gene (full-length DNA, cDNA or

- 10 046169 mRNA) encoding the protein FAD2, also known as FAD2-1, FAD2-1B or GMFAD2-1B. In one example, the FAD2 gene may be one or more genes selected from the group consisting of, but is not limited to, the following genes: genes encoding plant, e.g., soybean (Glycine max) FAD2 (e.g., under accession numbers NCBI NP_001341865.1, ХР_006605883.1, ХР_006605882.1, ХР_006605885.1, ХР_006605884.1 or

ХР_014627765.1), for example, FAD2 genes, presented under NCBI accession numbers NM_001354936.1, XM_006605820.2, ХМ_006605819.2, ХМ_006605822.2, ХМ_006605821.2 or

ХМ_014772279.1.

In any illustrative embodiment, the unsaturated fatty acid biosynthesis-associated factor may be FAD3.

The FAD3 (microsomal omega-3 fatty acid desaturase) gene refers to the gene (full-length DNA, cDNA or mRNA) encoding the FAD3 protein, also known as Fanx. In one example, the FAD3 gene may be one or more genes selected from the group consisting of, but is not limited to, the following genes: a gene encoding a plant, e.g., soybean (Glycine max) FAD3 (e.g., accession no. NCBI NP_001237507.1), for example, the FAD3 gene, presented under NCBI accession number NM_001250578.1.

In any illustrative embodiment, the unsaturated fatty acid biosynthesis-associated factor may be FAD6.

The FAD6 (fatty acid desaturase 6) gene refers to the gene (full-length DNA or mRNA) encoding the FAD6 protein, also known as FADC or SFD4. In one example, the FAD6 gene may be one or more genes selected from the group consisting of, but not limited to, the following genes: a gene encoding the plant, e.g., Arabidopsis thaliana FAD6 (e.g., NCBI accession number NP_194824. 1), for example, the FAD6 gene, presented under NCBI accession number NM_119243.4.

In any illustrative embodiment, the unsaturated fatty acid biosynthesis-associated factor may be FAD7.

The FAD7 (chloroplast omega-3 fatty acid desaturase isoform 2) gene refers to the gene (full-length DNA, cDNA or mRNA) encoding the FAD7 protein. In one example, the FAD7 gene may be one or more genes selected from the group consisting of, but is not limited to, the following genes: a gene encoding a plant, e.g., soybean (Glycine max) FAD7 (e.g., accession no. NCBI NP_001237361.1), for example, the FAD7 gene, presented under NCBI accession number NM_001250432.1.

In any illustrative embodiment, the unsaturated fatty acid biosynthesis-associated factor may be FAD8.

The FAD8 (chloroplast omega-3 fatty acid desaturase-like desaturase) gene refers to the gene (full-length DNA, cDNA or mRNA) encoding the FAD8 protein. In one example, the FAD8 gene may be one or more genes selected from the group consisting of, but is not limited to, the following genes: a gene encoding a plant, e.g., soybean (Glycine max) FAD8 (e.g., accession no. NCBI NP_001239777.1), for example, the FAD8 gene, presented under NCBI accession number NM_001252848.1.

The unsaturated fatty acid biosynthesis-associated factor can be obtained from a plant such as soybean, Arabidopsis thaliana, sesame, corn and the like. etc.

Gene information can be obtained from a well-known database such as the National Center for Biotechnology Information (NCBI) GeneBank.

In one illustrative embodiment of the present invention, the unsaturated fatty acid biosynthesis-associated factor, for example, FAD2, FAD3, FAD6, FAD7 or FAD8, may be an artificially manipulated unsaturated fatty acid biosynthesis-associated factor.

In a certain embodiment, the artificially manipulated unsaturated fatty acid biosynthesis-associated factor may be genetically manipulated.

Gene manipulation or modification can be accomplished by an artificial insertion, deletion, substitution, or inversion occurring in part or all of the genomic sequence of a wild-type gene. In addition, gene manipulation or modification can be achieved by merging the manipulation or modification of two or more genes.

For example, a gene may be further activated by such gene manipulation or modification such that the protein encoded by the gene to be expressed in protein form has an improved function over its natural function. As an example, if the function of the protein encoded by a particular gene is A, then the function of the protein expressed by the manipulated gene may be completely different from A or may include additional function (A+B) including A. For example, a fusion of two or more proteins may be expressed using two or more genes having different or complementary functions

- 11 046169 due to such genetic manipulation or modification.

For example, two or more proteins may be expressed separately or independently in cells using two or more genes having different or complementary functions due to such gene manipulation or modification.

The manipulated unsaturated fatty acid biosynthesis-associated factor can produce an unsaturated fatty acid by forming one or more double bonds in the saturated fatty acid.

The unsaturated fatty acid biosynthesis-associated factor, when manipulated, can initiate the formation of a new one or more double bonds in the unsaturated fatty acid.

The unsaturated fatty acid biosynthesis-associated factor, when manipulated, can change the positions of one or more double bonds included in the unsaturated fatty acid.

The manipulated unsaturated fatty acid biosynthesis-associated factor can initiate the removal of one or more double bonds in an unsaturated fatty acid having two or more double bonds.

The unsaturated fatty acid biosynthesis-associated factor, when manipulated, can initiate the conversion of a cis-unsaturated fatty acid to a trans-unsaturated fatty acid.

The unsaturated fatty acid biosynthesis-associated factor, when manipulated, can initiate the conversion of a trans-unsaturated fatty acid to a cis-unsaturated fatty acid.

Unsaturated fatty acid biosynthesis-associated factor, when manipulated, can control the amount of unsaturated fatty acid contained in a plant.

Unsaturated fatty acid biosynthesis-associated factor, when manipulated, can increase the content of a specific unsaturated fatty acid found in a plant.

Unsaturated fatty acid biosynthesis-associated factor, when manipulated, can reduce the content of a specific unsaturated fatty acid contained in a plant.

Manipulation includes all types of structural or functional modifications of the factor associated with the biosynthesis of unsaturated fatty acids.

Structural modification of the unsaturated fatty acid biosynthesis-associated factor includes all types of modifications that are not the same as wild-type modifications existing in nature.

For example, if the unsaturated fatty acid biosynthesis-associated factor is DNA, RNA or a gene, then the structural modification may be the loss of one or more nucleotides.

The structural modification may be the insertion of one or more nucleotides.

Here, the inserted nucleotides include all from the subject containing the unsaturated fatty acid biosynthesis associated factor and nucleotides supplied to the subject from outside.

The structural modification may be a substitution of one or more nucleotides.

The structural modification may be a chemical modification of one or more nucleotides.

In this case, chemical modification includes everything from the addition, removal, and replacement of chemical functional groups.

As another example, if the unsaturated fatty acid biosynthesis-associated factor is a peptide, polypeptide or protein, the structural modification may be a loss of one or more amino acids.

The structural modification may be the addition of one or more amino acids.

Here, the amino acids include everything from the subject containing an unsaturated fatty acid biosynthesis-associated factor and amino acids supplied to the subject from outside.

The structural modification may be a substitution of one or more amino acids.

The structural modification may be a chemical modification of one or more amino acids.

In this case, chemical modification includes everything from the addition, removal, and replacement of chemical functional groups.

The structural modification may be the partial or complete addition of another peptide, polypeptide or protein.

In this case, the other peptide, polypeptide or protein may be an unsaturated fatty acid biosynthesis-associated factor or a peptide, polypeptide or protein having

- 12 046169 another function.

The functional modification of the unsaturated fatty acid biosynthesis-associated factor may include all types having an improved or decreased function compared to that of the wild type, existing naturally, and having a third different function.

For example, if the unsaturated fatty acid biosynthesis-associated factor is a peptide, polypeptide or protein, then the functional modification may be a mutation in the unsaturated fatty acid biosynthesis-associated factor.

In this case, the mutation may be a mutation that enhances or suppresses the function of an unsaturated fatty acid biosynthesis-associated factor.

The functional modification may provide for the additional function of an unsaturated fatty acid biosynthesis-associated factor.

In this case, the additional function may be the same or a different function. In addition, an unsaturated fatty acid biosynthesis-associated factor having an additional function can be fused to another peptide, polypeptide or protein.

Functional modification may be an increase in functionality due to increased expression of an unsaturated fatty acid biosynthesis-associated factor.

Functional modification may be a decrease in functionality due to reduced expression of an unsaturated fatty acid biosynthesis-associated factor.

In an illustrative embodiment, the manipulated unsaturated fatty acid biosynthesis-associated factor can be induced by one or more of the following mutations:

complete or partial deletions of a factor associated with the biosynthesis of unsaturated fatty acids, that is, the gene to be manipulated (hereinafter referred to as the target gene), for example, a 1 bp deletion. or more nucleotides, e.g. 1-30, 1-27, 1-25, 1-23, 1-20, 1-15, 1-10, 1-5, 1-3 or 1 nucleotide in the target gene, substitution 1 By. or more nucleotides, for example, 1-30, 1-27, 1-25, 1-23, 1-20, 1-15, 1-10, 1-5, 1-3 or 1 nucleotide in the target gene, nucleotide, different from the wild type, and the insertion of one or more nucleotides, for example, 1-30, 1-27, 1-25, 1-23, 1-20, 1-15, 1-10, 1-5, 1-3 or 1 nucleotide (each independently selected from A, T, C and G), at a specific location in the target gene.

Part of the modified target gene (target region) may be continuous from 1 bp. or more, 3 p.o. or more, 5 p.o. or more, 7 bp or more, 10 bp or more, 12 bp or more, 15 bp or more, 17 bp or more, or 20 bp or more, for example, 1 p.o. - 30 bp, 3 bp - 30 bp, 5 bp - 30 bp, 7 bp - 30 bp, 10 bp -30 bp, 12 bp - 30 bp, 15 bp - 30 bp, 17 bp - 30 bp, 20 bp - 30 p.o., 1 p.o. - 27 bp, 3 bp - 27 bp, 5 bp - 27 bp, 7 bp - 27 bp, 10 bp - 27 bp, 12 bp 27 bp, 15 bp - 27 bp, 17 bp - 27 bp, 20 bp - 27 p.o., 1 p.o. - 25 bp, 3 bp - 25 bp, 5 bp - 25 bp, 7 bp - 25 bp, 10 bp - 25 bp, 12 bp - 25 bp, 15 bp - 25 bp, 17 bp - 25 bp, 20 bp - 25 p.o., 1 p.o. - 23 bp, 3 bp - 23 bp, 5 bp - 23 bp, 7 bp - 23 bp, 10 bp - 23 bp, 12 bp - 23 bp, 15 bp - 23 bp, 17 bp 23 bp, 20 bp - 23 p.o., 1 p.o. - 20 bp, 3 bp - 20 bp, 5 bp - 20 bp, 7 bp - 20 bp, 10 bp - 20 bp, 12 bp - 20 bp, 15 bp - 20 bp, 17 bp - 20 bp, 21 bp - 25 bp, 18 bp - 22 bp, or 21 bp - 23 bp, part of the main sequence of the gene.

A system designed to control unsaturated fatty acids.

One aspect of the present invention relates to an unsaturated fatty acid control system that controls unsaturated fatty acid biosynthesis by artificially manipulating an unsaturated fatty acid biosynthesis-associated factor.

The term unsaturated fatty acid control system as used herein includes all phenomena that have the effect of promoting or inhibiting unsaturated fatty acid biosynthesis and/or increasing or inhibiting unsaturated fatty acid production by altering the functions of an unsaturated fatty acid biosynthesis-associated factor , subjected to artificial manipulation, and also includes materials, compositions, methods and applications directly or indirectly involved in the control system of unsaturated fatty acid biosynthesis.

Each factor that makes up a system designed to provide control of unsaturated fatty acid biosynthesis is also called an unsaturated fatty acid control factor.

The system in accordance with the present invention contains a modified mechanism in the plant organism, which is associated with an artificially manipulated factor associated with the biosynthesis of unsaturated fatty acids. By using an artificially manipulated unsaturated fatty acid biosynthesis associated factor, in any illustrative embodiment, the biosynthesis of a C8-24:D1 unsaturated fatty acid can be controlled,

- 13 046169 In any illustrative embodiment, the biosynthesis of a C8-24:D2 unsaturated fatty acid can be controlled, in any illustrative embodiment, the amount of C8-24:D1 unsaturated fatty acid produced can be controlled, in any illustrative embodiment, the amount of C8-24 produced can be controlled :D2 unsaturated fatty acid, in any illustrative embodiment, the C8-24:D1 unsaturated fatty acid content of a plant body can be controlled, in any illustrative embodiment, the C8-24:D2 unsaturated fatty acid content of a plant body can be controlled, in any illustrative embodiment implementation, the ratio of C8-24:D1 unsaturated fatty acid to C8-24:D2 unsaturated fatty acid in a plant organism can be controlled, in any illustrative embodiment, a double bond in the C8-24:D1 unsaturated fatty acid can be added or removed, and in any In an exemplary embodiment, a double bond may be added or removed from the C8-24:D2 unsaturated fatty acid.

In another illustrative embodiment, a system for controlling unsaturated fatty acid in accordance with the present invention includes a composition for manipulating an unsaturated fatty acid biosynthesis-associated factor.

The manipulation composition may be a composition capable of artificially manipulating an unsaturated fatty acid biosynthesis-associated factor, and preferably a composition for gene manipulation.

Next, a composition intended for gene manipulation will be described.

A composition intended for manipulation of a factor associated with the biosynthesis of unsaturated fatty acids.

Manipulation or modification of substances involved in the unsaturated fatty acid biosynthesis-associated factor and unsaturated fatty acid control system of the present invention is preferably accomplished through genetic manipulation.

In one aspect, a composition and method may be provided for manipulating a genome by targeting a partial or complete non-coding or coding region of an unsaturated fatty acid biosynthesis-associated factor.

In an illustrative embodiment, the composition and method can be used to manipulate or modify one or more unsaturated fatty acid biosynthesis-associated genes involved in the formation of a desired unsaturated fatty acid control system. Manipulation or modification can be accomplished by modifying the nucleic acids that make up a gene. As a result of the manipulation, everything from knockdown, knockout and knockout is included.

In an illustrative embodiment, the manipulation can be accomplished by targeting a promoter region or transcription sequence, such as an intronic or exonic sequence. The coding sequence, eg the coding region, the original coding region, can be targeted for modification by expression and knockout.

In an illustrative embodiment, the modification of nucleic acids may be the substitution, deletion and/or insertion of one or more nucleotides, for example, 1-30 bp, 1-27 bp, 1-25 bp, 1- 23 bp, 1-20 bp, 1-15 bp, 1-10 bp, 1-5 bp, 1-3 bp. or 1 p.o. nucleotide.

In an illustrative embodiment, to knock out one or more unsaturated fatty acid biosynthesis-associated genes, eliminate expression of one or more genes, or one or more knockouts of one or two alleles, the above-mentioned region may be targeted such that one or more unsaturated fatty acid biosynthesis-associated genes will contain a deletion or mutation.

In an illustrative embodiment, gene knockdown can be used to reduce the expression of undesirable alleles or transcriptomes.

In an illustrative embodiment, non-coding sequences of a promoter, enhancer, intron, 3'UTR, and/or polyadenylation signal may be targeted for use in modifying an unsaturated fatty acid biosynthesis-associated gene affecting unsaturated fatty acid biosynthesis function.

In an illustrative embodiment, the activity of an unsaturated fatty acid biosynthesis-associated gene can be regulated, for example, activated or inactivated, by modifying the nucleic acid of the gene.

In an illustrative embodiment, modification of a gene's nucleic acid may initiate catalytic cleavage of the single strand or double strands, i.e., disrupt the strands

- 14 046169 nucleic acid in a specific region of the target gene with the help of a guide nucleic acid-editing protein complex, which leads to inactivation of the target gene.

In an illustrative embodiment, breaks in the nucleic acid strand can be repaired through a mechanism such as homologous recombination or non-homologous end joining (NHEJ).

In this case, if the NHEJ mechanism occurs, a change in the DNA sequence at the cleavage site is induced, which leads to gene inactivation. NHEJ repair can induce replacement, insertion, or deletion of a short gene fragment and can be used to induce knockout of the corresponding gene.

In another aspect, the present invention provides a composition for manipulating an unsaturated fatty acid biosynthesis-associated factor.

A composition for manipulation is one that is capable of artificially manipulating an unsaturated fatty acid biosynthesis-associated factor, and preferably a composition for gene manipulation.

The composition can be used in gene manipulation of one or more unsaturated fatty acid biosynthesis-associated factors involved in the formation of the desired system for controlling the unsaturated fatty acid.

Gene manipulation can be performed taking into account the process of regulation of gene expression.

In an illustrative embodiment, this can be accomplished by selecting appropriate means to manipulate each step of transcription, RNA processing, RNA transport, RNA cleavage, translation, and protein modification regulation steps.

In an illustrative embodiment, small RNA (sRNA) interacts with or reduces the stability of mRNA using RNA interference (RNAi) or RNA silencing, and in some cases cleaves the mRNA, interrupting the delivery of protein synthesis information, resulting in regulation of the expression of genetic information .

Gene manipulation can be accomplished by modifying the nucleic acids that make up the unsaturated fatty acid biosynthesis-associated factor. As a result of the manipulation, everything from knockdown, knockout and knockout is included.

In a particular embodiment, the modification of nucleic acids may be a substitution, deletion and/or insertion of one or more nucleotides, for example, 1-30 bp, 1-27 bp, 1-25 bp, 1- 23 bp, 1-20 bp, 1-15 bp, 1-10 bp, 1-5 bp, 1-3 bp. or 1 p.o. nucleotide.

In a certain embodiment, to knock out one or more unsaturated fatty acid biosynthesis-associated factors, to eliminate the expression of one or more factors, or for one or more knockouts of one or two alleles, a gene can be manipulated such that one or more unsaturated fatty acid biosynthesis-associated factors acid factors will contain a deletion or mutation.

In a certain embodiment, knockdown of an unsaturated fatty acid biosynthesis-associated factor can be used to reduce the expression of undesirable alleles or transcriptomes.

In a certain embodiment, the modification of nucleic acids may be the insertion of one or more nucleic acid fragments or genes. Here, the nucleic acid fragment may be a nucleic acid sequence consisting of one or more nucleotides, and the length of the nucleic acid fragment may be 1-40 bp, 1-50 bp, 160 bp, 1- 70 bp, 1-80 bp, 1-90 bp, 1-100 bp, 1-500 bp or 1-1000 bp. In this case, the inserted gene may be one of the factors associated with the biosynthesis of unsaturated fatty acids or a gene with a different function.

In an exemplary embodiment, the modification of nucleic acids may use a wild-type enzyme or a variant of an enzyme that is capable of catalytically hydrolyzing (breaking down) the bonds between nucleic acids in a DNA or RNA molecule, preferably a DNA molecule. A guide nucleic acid-editing protein complex can also be used.

For example, a gene can be manipulated using one or more nucleases selected from the group consisting of meganuclease, zinc finger nuclease, CRISPR/Cas9 (Cas9 protein), CRISPR-Cpf1 (Cpf1 protein) and TALE nuclease, thereby achieving regulation of the expression of genetic information.

In a certain embodiment, without limitation, gene manipulation may be mediated by NHEJ or homology-directed repair (HDR) using a guide nucleic acid-editing protein complex, such as the CRISPR/Cas system.

In the case of the NHEJ mechanism, a change in the DNA sequence at the cleavage site can be induced, thereby inactivating the gene. NHEJ repair can induce replacement, insertion or deletion of a short gene fragment and can be used to induce knockout of the corresponding gene.

- 15 046169

In another aspect, the present invention may provide a gene manipulation site.

In an illustrative embodiment, if a gene is modified by NHEJ-mediated modification, the site for gene manipulation may be a site in the gene that initiates the reduction or elimination of expression of an unsaturated fatty acid biosynthesis-associated gene product.

For example, the site may be located in an original coding region, a promoter sequence, an enhancer sequence, a specific intronic sequence, or a specific exonic sequence.

In an illustrative embodiment, a composition for manipulating an unsaturated fatty acid biosynthesis-associated factor may target an unsaturated fatty acid biosynthesis-associated factor that influences the regulation of unsaturated fatty acid biosynthesis, such as the FAD gene, preferably the FAD2 gene, the FAD3 gene, the FAD6, FAD7 gene or FAD8 gene serving as the subject of manipulation. Most preferably, a composition for manipulating an unsaturated fatty acid biosynthesis-associated factor may target the FAD2 gene as the target for manipulation.

Examples of target regions of the FAD2 gene, that is, target sequences for regions in which gene manipulation is carried out or which are recognized for gene manipulation, are briefly described in table. 1.

The target sequence may target one or more genes.

The target sequence can simultaneously target two or more genes. Here, the two or more genes may be homologous genes or heterologous genes.

A gene may contain one or more target sequences.

A gene can be targeted simultaneously by two or more target sequences.

The gene can be changed by site and by the number of gene manipulations according to the number of target sequences.

Gene manipulation can be designed in various forms depending on the number and positions of the target sequences.

Gene manipulation can occur simultaneously in two or more target sequences. Here, two or more target sequences may be present in a homologous gene or a heterologous gene.

Gene manipulation can be carried out simultaneously on two or more genes. Here, the two or more genes may be homologous genes or heterologous genes.

The following tables further show examples of target sequences that may be used in embodiments of the present invention.

Table 1

FAD2 gene target sequences

No. Target sequence (including RAM) 1 ATAGATTGGCCATGCAATGAGGG (SEQ ID NO: 1) 2 AATAGATTGGCCATGCAATGAGG (SEQ ID NO: 2) 3 CCTTGGAGAACCCAATAGATTGG (SEQ ID NO: 3) 4 TGGGTGATTGCTCACGAGTGTGG (SEQ ID NO: 4) 5 TTTTAGTCCCTTATTTCTCATGG (SEQ ID NO: 5) 6 AAACACTTCATCACGGTCAAGGG (SEQ ID NO: 6) 7 GTGTTTGGAACCCTTGAGAGAGG (SEQ ID NO: 7) 8 GTGAATGGTGGCTTTGTGTTTGG (SEQ ID NO: 8) 9 ACAAAGCCACCATTCACTGTTGG (SEQ ID NO: 9) 10 AGTTGGCCAACAGTGAATGGTGG (SEQ ID NO: 10) eleven TTGAGTTGGCCAACAGTGAATGG (SEQ ID NO: 11) 12 TGAAAGGTCATAAACAACATAGG (SEQ ID NO: 12) 13 CAAACACTTCATCACGGTCAAGG (SEQ ID NO: 13) 14 AACCAAAATCCAAAGTTGCATGG (SEQ ID NO: 14) 15 TGGGAGCATAAGGGTGGTAGTGG (SEQ ID NO: 15) 16 AATATATGGGGAGCATAAGGGTGG (SEQ Ш NO: 16) 17 GTTTGGCTGCTATGTGTTTATGG (SEQ Ш NO: 17) 18 TTTGGCTGCTATGTGTTTATGGG (SEQ ID NO: 18) 19 TTGGCTGCTATGTGTTTATGGG (SEQ ID NO: 19) 20 GCAACTATTGGACAGAGATTATGG (SEQ ID NO: 20) 21 CACCATTTTACAAGGCACTGTGG (SEQ ID NO: 21) 22 CTTCATCTGGCTCCACATAGAGG (SEQ Ш NO: 22) 23 CTCTATGTGGAGCCAGATGAAGG (SEQ ID NO: 23) 24 TTCTCGGATGTTCCTTCATCTGG (SEQ ID NO: 24) 25 AGATGAAGGAACATCCGAGAAGG (SEQ ID NO: 25)

- 16 046169

26 GATGAAGGAACATCCGAGAAGGG (SEQ ID NO: 26) 27 CATCCGAGAAGGGCGTGTATTGG (SEQ ID NO: 27) 28 GTACCAATACACGCCCTTCTCGG (SEQ ID NO: 28) 29 AGAAGGGCGTGTATTGGTACAGG (SEQ Ш NO: 29) thirty TTGGGACAAACACTTCATCACGG (SEQ ID NO: 30)

Composition for manipulation - gene scissors system.

A system for controlling an unsaturated fatty acid in accordance with the present invention may include a guide nucleic acid-editing protein complex as a composition for manipulating an unsaturated fatty acid biosynthesis-associated factor.

Guide nucleic acid-editing protein complex.

The term guide nucleic acid-editing protein complex refers to a complex formed by the interaction between a guide nucleic acid and an editing protein, and a nucleic acid-protein complex includes a guide nucleic acid and an editing protein.

The term targeting nucleic acid refers to a nucleic acid capable of recognizing a target nucleic acid, gene, chromosome or protein.

The guide nucleic acid may be in the form of DNA, RNA, or a DNA/RNA hybrid and may have a nucleic acid sequence of 5-150 bases.

The guide nucleic acid may include one or more domains.

The domains may be, without limitation, a targeting domain, a first complementary domain, a linker domain, a second complementary domain, a proximal domain, or a tail domain.

The guide nucleic acid may include two or more domains, which may be repeats of the same domains or may be different domains.

The guide nucleic acid may have one contiguous nucleic acid sequence.

For example, one contiguous nucleic acid sequence may be (N)m, where N is A, T, C, or G, or A, U, C, or G, and m is an integer from 1 to 150.

The guide nucleic acid may have two or more contiguous nucleic acid sequences.

For example, two or more contiguous nucleic acid sequences may be (N) _m and (N)o, where N is A, T, C or G, or A, U, C or G, m and o are an integer from 1 to 150, and m and o may be the same or different from each other.

The term editing protein refers to a peptide whose polypeptide or protein can directly bind or interact with a nucleic acid without directly binding to it.

The editing protein may be an enzyme. The editing protein may be a fusion protein.

As used herein, the term fusion protein refers to a protein that is produced by fusing an enzyme with an additional domain, peptide, polypeptide or protein.

The term enzyme refers to a protein that contains a domain capable of degrading a nucleic acid, gene, chromosome or protein.

The additional domain, peptide, polypeptide or protein may be a functional domain, peptide, polypeptide or protein that has the same or different function than the enzyme.

The fusion protein may include an additional domain, peptide, polypeptide or protein at or near one or more amino-terminal (N-terminal) regions of the enzyme; carboxy-terminal (C-terminal) or near it; the middle part of the enzyme and their combinations.

The fusion protein may include a functional domain, peptide, polypeptide or protein at or near one or more regions of the N-terminus of the enzyme; C-end or near it; the middle part of the enzyme and their combinations.

The guide nucleic acid-editing protein complex may serve to modify the subject.

The subject may be a target nucleic acid, gene, chromosome or protein.

For example, a targeting nucleic acid-editing protein complex may result in the resulting regulation (eg, inhibition, suppression, reduction, enhancement, or stimulation) of the expression of a protein of interest, the removal of a protein, or the expression of a new protein.

In this case, the guiding nucleic acid-editing protein complex can act at the level of DNA, RNA, gene or chromosome.

The guiding nucleic acid-editing protein complex can act at the stages of gene transcription and translation.

- 17 046169

The guide nucleic acid-editing protein complex can act at the protein level.

1. Guide nucleic acids.

A guide nucleic acid is a nucleic acid that is capable of recognizing a target nucleic acid, gene, chromosome or protein and forms a guide nucleic acid-protein complex.

Here, the guide nucleic acid is designed to recognize or target the nucleic acid, gene, chromosome or protein that is targeted by the guide nucleic acid-protein complex.

The guide nucleic acid may be in the form of DNA, RNA or a mixture of DNA/RNA and has a nucleic acid sequence of 5-150 bases.

The guide nucleic acid may be present in linear or circular form.

The guide nucleic acid may be a single contiguous nucleic acid sequence.

For example, one contiguous nucleic acid sequence may be (N) _m , where N is A, T, C, or G, or A, U, C, or G, and m is an integer from 1 to 150.

The guide nucleic acid may be two or more contiguous nucleic acid sequences.

For example, two or more contiguous nucleic acid sequences may be (N) _m and (N)o, where N is A, T, C or G, or A, U, C or G, m and o are an integer from 1 to 150, and may be the same or different from each other.

The guide nucleic acid may include one or more domains.

In this case, the domains may be, without limitation, a targeting domain, a first complementary domain, a linker domain, a second complementary domain, a proximal domain, or a tail domain.

The guide nucleic acid may include two or more domains, which may be repeats of the same domains or may be different domains.

The domains will be described below.

i) Directing domain.

The term targeting domain is a domain with a complementary targeting sequence that is capable of forming a complementary bond with a target sequence in a target gene or nucleic acid, and serves to specifically interact with the target gene or nucleic acid.

A guide sequence is a nucleic acid sequence complementary to a target sequence in a target gene or nucleic acid that is, for example, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more complementary or fully complementary.

The targeting domain may be a sequence of 5-50 bases.

By way of example, the targeting domain may be a sequence of 5-50, 10-50, 15-50, 20-50, 25-50, 30-50, 35-50, 40-50, or 45-50 bases.

As another example, the targeting domain may be a sequence of 1-5, 5-10, 10-15, 15-20, 20-25, 25-30, 30-35, 35-40, 40-45, or 45- 50 bases.

The guide domain may have a guide sequence.

The guide sequence may be a complementary base sequence that can form a complementary linkage to a target sequence in the target gene or nucleic acid.

The guide sequence may be a nucleic acid sequence complementary to a target sequence in the target gene or nucleic acid that is, for example, at least 70%, 75%, 80%, 85%, 90%, 95% or greater complementarity or complete complementarity .

The guide sequence may be a sequence of 5-50 bases.

By way of example, the targeting domain may be a sequence of 5-50, 10-50, 15-50, 20-50, 25-50, 30-50, 35-50, 40-50, or 45-50 bases.

As another example, the guide sequence may be a sequence of 1-5, 5-10, 10-15, 15-20, 20-25, 25-30, 30-35, 35-40, 40-45, or 45-50 grounds.

In addition, the targeting domain may include a targeting sequence and an additional base sequence.

Additional base sequence can be used to improve or reduce the function of the targeting domain.

Additional base sequence can be used to improve or reduce the function of the guide sequence.

- 18 046169

The additional base sequence may be a sequence of 1-35 bases.

In one example, the additional base sequence may be a sequence of 5-35, 10-35, 15-35, 20-35, 25-35, or 30-35 bases.

As another example, the additional base sequence may be a sequence of 1-5, 5-10, 10-15, 15-20, 20-25, 25-30, or 30-35 bases.

An additional base sequence may be located at the 5' end of the guide sequence.

An additional base sequence may be located at the 3' end of the guide sequence.

ii) First complementary domain.

The term first complementary domain is a nucleic acid sequence comprising a nucleic acid sequence complementary to the second complementary domain and having a sufficient degree of complementarity to form a double strand with the second complementary domain.

The first complementary domain may be a sequence of 5-35 bases.

By way of example, the first complementary domain may be a sequence of 5-35, 10-35, 15-35, 20-35, 25-35, or 30-35 bases.

As another example, the first complementary domain may be a sequence of 1-5, 5-10, 10-15, 15-20, 20-25, 25-30, or 30-35 bases.

iii) Linker domain.

The term linker domain is a nucleic acid sequence connecting two or more domains, which are two or more identical or different domains. A linker domain may be connected to two or more domains by a covalent bond or a non-covalent bond, or may connect two or more domains by a covalent bond or a non-covalent bond.

The linker domain may be a sequence of 1-30 bases.

In one example, the linker domain may be a sequence of 1-5, 5-10, 1015, 15-20, 20-25, or 25-30 bases.

As another example, the linker domain may be a sequence of 130, 5-30, 10-30, 15-30, 20-30, or 25-30 bases.

iv) Second complementary domain.

The term second complementary domain is a nucleic acid sequence comprising a nucleic acid sequence complementary to the first complementary domain and having a sufficient degree of complementarity to form a double strand with the first complementary domain.

The second complementary domain may have a base sequence complementary to the first complementary domain and a base sequence that is not complementary to the first complementary domain, e.g., a base sequence that does not form a double strand with the first complementary domain, and may have a longer base sequence than the first complementary domain. domain.

The second complementary domain may have a sequence of 5-35 bases.

By way of example, the second complementary domain may be a sequence of 1-5, 35-10, 35-15, 35-20, 35-25, 35-30, or 35-35 bases.

As another example, the second complementary domain may be a sequence of 1-5, 5-10, 10-15, 15-20, 20-25, 25-30, or 30-35 bases.

v) Proximal domain.

The term proximal domain is a nucleic acid sequence located adjacent to the second complementary domain.

The proximal domain may have a complementary base sequence and may be formed as a double strand by complementary base sequence.

The proximal domain may be a sequence of 1-20 bases.

In one example, the proximal domain may be a sequence of 1-20, 520, 10-20, or 15-20 bases.

As another example, the proximal domain may be a sequence of 1-5, 5-10, 10-15 or 15-20 bases.

vi) Tail domain.

The term tail domain is a nucleic acid sequence located at one or more ends at both ends of the guide nucleic acid.

The tail domain may have a complementary base sequence and may be formed as a double strand by complementary base sequence.

The tail domain can be a sequence of 1-50 bases.

- 19 046169

By way of example, the tail domain may be a sequence of 5-50, 10-50, 15-50, 20-50, 25-50, 30-50, 35-50, 40-50, or 45-50 bases.

As another example, the tail domain may be a sequence of 1-5, 5-10, 10-15, 15-20, 20-25, 25-30, 30-35, 35-40, 40-45, or 45-50 grounds.

In this case, part or all of the nucleic acid sequences are included in domains, that is, the targeting domain, the first complementary domain, the linker domain, the second complementary domain, the proximal domain and the tail domain may selectively or additionally contain a chemical modification.

The chemical modification may be, but is not limited to, methylation, acetylation, phosphorylation, phosphorothioate linkage, locked nucleic acid (LNA), 2'-O-methyl3'-phosphorothioate (MS) or 2'-O-methyl-3'-thioPACE (MSP) .

The guide nucleic acid includes one or more domains. The guide nucleic acid may include a targeting domain. The guide nucleic acid may include a first complementary domain. The guide nucleic acid may include a linker domain. The guide nucleic acid may include a second complementary domain. The guide nucleic acid may include a proximal domain. The guide nucleic acid may include a tail domain. In this case, it could be 1, 2, 3, 4, 5, 6 or more domains.

The guide nucleic acid may include 1, 2, 3, 4, 5, 6 or more guide domains.

The guide nucleic acid may include 1, 2, 3, 4, 5, 6 or more first complementary domains.

The guide nucleic acid may include 1, 2, 3, 4, 5, 6 or more linker domains.

The guide nucleic acid may include 1, 2, 3, 4, 5, 6 or more second complementary domains.

The guide nucleic acid may include 1, 2, 3, 4, 5, 6 or more proximal domains.

The guide nucleic acid may include 1, 2, 3, 4, 5, 6 or more tail domains.

In this case, one type of domain may be duplicated in the guide nucleic acid.

The guide nucleic acid may include multiple domains, with or without duplication.

The guide nucleic acid may include the same type of domain. Here, the same type of domain may have the same nucleic acid sequence or different nucleic acid sequences.

The guide nucleic acid may include two types of domains. In this case, two different types of domains may have different nucleic acid sequences or the same nucleic acid sequence.

The guide nucleic acid may include three types of domains. In this case, the three different types of domains may have different nucleic acid sequences or the same nucleic acid sequence.

The guide nucleic acid may include four types of domains. In this case, the four different types of domains may have different nucleic acid sequences or the same nucleic acid sequence.

The guide nucleic acid may include five types of domains. In this case, the five different types of domains may have different nucleic acid sequences or the same nucleic acid sequence.

The guide nucleic acid may include six types of domains. In this case, the six different types of domains may have different nucleic acid sequences or the same nucleic acid sequence.

For example, a targeting nucleic acid may consist of [targeting domain][first complementary domain]-[linker domain]-[second complementary domain][linker domain]-[targeting domain]-[first complementary domain]-[linker domain]-[ second complementary domain]. In this case, the two targeting domains may include targeting sequences for different or the same purposes, wherein the first two complementary domains and the two second complementary domains may have the same or different nucleic acid sequences. If the targeting domains include targeting sequences for different targets, then the targeting nucleic acids can specifically bind to two different targets, in which case the specific bindings can be performed simultaneously or sequentially. In addition, linker domains can be cleaved by specific enzymes, and guide nucleic acids can be separated into two or three parts in the presence of specific enzymes.

As a specific example of a guide nucleic acid according to the present

- 20 046169 gRNA will be described below by the invention.

The term gRNA refers to a nucleic acid capable of specifically targeting the gRNA-CRISPR enzyme complex, that is, the CRISPR complex, to a target gene or nucleic acid. In addition, gRNA is a nucleic acid-specific RNA that can bind to the CRISPR enzyme and direct the CRISPR enzyme to a target gene or nucleic acid.

gRNA can contain multiple domains. Due to each domain, interactions can occur within the three-dimensional structure or active form of the gRNA strand, or between these strands.

gRNA can be called a single-stranded gRNA (a single RNA molecule) or a double-stranded gRNA (comprising more than one, usually two separate RNA molecules).

In one illustrative embodiment, a single-stranded gRNA may include a targeting domain, that is, a domain including a targeting sequence capable of forming a complementary linkage to a target gene or nucleic acid; first complementary domain; linker domain; a second complementary domain, a domain having a sequence complementary to the sequence of the first complementary domain, thereby forming a double-stranded nucleic acid with a first complementary domain; a proximal domain and optionally a tail domain in the 5'-3' direction.

In another embodiment, the double-stranded gRNA may include a first strand that includes a targeting domain, that is, a domain including a targeting sequence capable of forming a complementary linkage to a target gene or nucleic acid and a first complementary domain; and a second strand that includes a second complementary domain, a domain having a sequence complementary to that of the first complementary domain, thereby forming a double-stranded nucleic acid with a first complementary domain; a proximal domain and optionally a tail domain in the 5'-3' direction.

In this case, the first strand may be called crRNA and the second strand may be called tracrRNA. The crRNA may include a guide domain and a first complementary domain, and the tracrRNA may include a second complementary domain, a proximal domain, and optionally a tail domain.

In a further embodiment, the single-stranded gRNA may include a targeting domain, that is, a domain including a targeting sequence capable of forming a complementary linkage to a target gene or nucleic acid; first complementary domain; a second complementary domain and a domain having a sequence complementary to the sequence of the first complementary domain, thereby forming a double-stranded nucleic acid with the first complementary domain in the 5'-3' direction.

i) Directing domain.

The targeting domain includes a complementary targeting sequence capable of forming a complementary linkage to a target sequence in the target gene or nucleic acid. The guide sequence may be a nucleic acid sequence that has complementarity with a target sequence in the target gene or nucleic acid, for example, at least 70%, 75%, 80%, 85%, 90% or 95% complementarity or greater complementarity or complete complementarity . The targeting domain is believed to allow the gRNA-Cas complex, that is, the CRISPR complex, to specifically interact with a target gene or nucleic acid.

The targeting domain may be a sequence of 5-50 bases.

In an illustrative embodiment, the targeting domain may be a sequence of 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 bases.

In an illustrative embodiment, the targeting domain may comprise a sequence of 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 bases.

In this case, the targeting domain may include a targeting sequence.

The guide sequence may be a complementary base sequence capable of forming a complementary linkage to a target sequence in the target gene or nucleic acid.

The guide sequence may be a nucleic acid sequence complementary to a target sequence in the target gene or nucleic acid that is, for example, at least 70%, 75%, 80%, 85%, 90% or 95% or greater complementarity or complete complementarity .

In one illustrative embodiment, the targeting sequence may be a nucleic acid sequence complementary to a target gene, that is, a target sequence of an unsaturated fatty acid biosynthesis-associated factor such as a FAD gene, preferably a FAD2 gene, a FAD3 gene, a FAD6 gene, a FAD7 gene, or a FAD8 gene , which is characterized, for example, by at least 70%, 75%, 80%, 85%, 90% or 95%, or more complementarity or complete complementarity.

The guide sequence may be a sequence of 5-50 bases

- 21 046169 ni.

In an illustrative embodiment, the guide sequence may be a sequence of 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 bases.

In one illustrative embodiment, the guide sequence may include a sequence of 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 bases.

In one illustrative embodiment, the targeting sequence may be a nucleic acid sequence complementary to the FAD2 gene target sequence, which is a sequence of 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 bases.

In one illustrative embodiment, the targeting sequence may be a nucleic acid sequence complementary to the FAD3 gene target sequence, which is a sequence of 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 bases.

In one illustrative embodiment, the targeting sequence may be a nucleic acid sequence complementary to the FAD6 gene target sequence, which is a sequence of 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 bases.

In one illustrative embodiment, the targeting sequence may be a nucleic acid sequence complementary to the FAD7 gene target sequence, which is a sequence of 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 bases.

In one illustrative embodiment, the targeting sequence may be a nucleic acid sequence complementary to the FAD8 gene target sequence, which is a sequence of 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 bases.

In this case, the target sequences of the target genes, that is, factors associated with the biosynthesis of unsaturated fatty acids, such as the FAD2 gene, for the guide sequence are given above in table. 1, but the present invention is not limited to them.

In this case, the targeting domain may include a targeting sequence and an additional base sequence.

The additional base sequence may be a sequence of 1-35 bases.

In one illustrative embodiment, the additional base sequence may be a sequence of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 bases.

For example, the additional base sequence may be a single guanine (G) base sequence, or a double GG base sequence.

An additional base sequence may be located at the 5' end of the guide sequence.

An additional base sequence may be located at the 3' end of the guide sequence.

In particular, part or all of the base sequences of the targeting domain may include chemical modification. The chemical modification may be methylation, acetylation, phosphorylation, phosphorothioate linkage, locked nucleic acid (LNA), 2'O-methyl-3'-phosphorothioate (MS) or 2'-O-methyl-3'-thioPACE (MSP), however, the present invention is not limited to them.

ii) First complementary domain.

The first complementary domain includes a nucleic acid sequence complementary to the second complementary domain and having a sufficient degree of complementarity to form a double strand with the second complementary domain.

In this case, the first complementary domain may be a sequence of 5-35 bases. The first complementary domain may include a sequence of 5-35 bases.

In one illustrative embodiment, the first complementary domain may be the sequence of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 , 24 or 25 bases.

In another embodiment, the first complementary domain may include the sequence of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 bases.

The first complementary domain may have homology to a naturally occurring first complementary domain or may be derived from a naturally occurring first complementary domain. In addition, the first complementary domain may have a difference in the base sequence of the first complementary domain according to species existing in nature, may be derived from the first complementary domain contained in species existing in nature, or

- 22 046169 may be characterized by partial or complete homology with the first complementary domain contained in species existing in nature.

In one illustrative embodiment, the first complementary domain may have partial, i.e., at least 50% or greater or complete homology with the first complementary domain of Streptococcus pyogenes, Campylobacter jejuni, Streptococcus thermophilus, Staphylococcus aureus or Neisseria meningitides, or with the first complementary domain derived out of him.

For example, if the first complementary domain is the first complementary domain of Streptococcus pyogenes or a first complementary domain derived therefrom, then the first complementary domain may be 5'-GUUUUAGAGCUA-3' (SEQ ID NO: 42) or a base sequence characterized by partial, that is, at least 50% or greater or complete homology with 5'-GUUUUAGAGCUA-3' (SEQ ID NO: 42).

In this case, the first complementary domain may further include (X) _n , resulting in 5'-GUUUUAGAGCUA(X) _n -3' (SEQ ID NO: 42). χ may be selected from the group consisting of bases A, T, U, and G, and n may be a number of bases that is an integer between 5 and 15. In this case, (X) _n may have n repeats of the same bases or combination of n bases A, T, U and G.

In another embodiment, if the first complementary domain is the first complementary domain of Campylobacter jejuni or a first complementary domain derived therefrom, then the first complementary domain may be 5'-GUUUUAGUCCCULnJUUAAAUUUCUU-3' (SEQ ID NO: 43) or a base sequence characterized by partial, that is, at least 50% or greater or complete homology with 5'-GUUUUAGUCCCUUUUUAAAUUUCUU-3' (SEQ ID NO: 43).

In this case, the first complementary domain may further include (X) _n , resulting in 5NGUUUUAGUCCCUUUUUAAAUUUCUU(X) _n -3 (SEQ ID NO: 43). χ may be selected from the group consisting of bases A, T, U, and G, and n may be a number of bases that is an integer between 5 and 15. In this case, (X) _n may have n repeats of the same bases or a combination of n bases A, T, U and G.

In another embodiment, the first complementary domain may have partial, i.e., at least 50% or greater or complete homology with the first complementary domain

Parcubacteria bacterium (GWC2011_GWC2_44_17), Lachnospiraceae bacterium (MC2017), Butyrivibrio proteoclasiicus, Peregrinibacteria bacterium (GW2011_GWA_33_10), Acidaminococcus sp.

(BV3L6), Porphyromonas macacae, Lachnospiraceae bacterium (ND2006), Porphyromonas crevioricanis, Prevotella disiens, Moraxella bovoculi (237), Smiihella sp. (SC KO8D17), Leptospira inadai, Lachnospiraceae bacterium (MA2020), Francisella novicida (UI 12), Candidatus Methanoplasma termitum or Eubacterium eh gens.

or the first complementary domain derived from them.

For example, if the first complementary domain is the first complementary domain of Parcubacteria bacterium or a first complementary domain derived therefrom, then the first complementary domain may be 5'-UUUGUAGAU-3' or a base sequence characterized by partial, i.e., at least 50% or greater homology with 5'-UUUGUAGAU-3'.

In this case, the first complementary domain may further include (X) _n , resulting in 5' -(X) _n UUUGUAGAU-3'.

X may be selected from the group consisting of bases A, T, U and G, and n may be a number of bases that is an integer from 1 to 5. In this case, (X)n may have n repetitions of the same bases or combination of n bases A, T, U and G.

In particular, some or all of the base sequences of the first complementary domain may have a chemical modification. The chemical modification may be methylation, acetylation, phosphorylation, phosphorothioate linkage, locked nucleic acid (LNA), 2'-O-methyl-3'-phosphorothioate (MS) or 2'-O-methyl-3'-thioPACE (MSP) , but the present invention is not limited to them.

iii) Linker domain.

A linker domain is a nucleic acid sequence connecting two or more domains, which are two or more identical or different domains. A linker domain may be connected to two or more domains by a covalent bond or a non-covalent bond, or may connect two or more domains by a covalent or non-covalent bond.

- 23 046169

The linker domain may be a nucleic acid sequence connecting the first complementary domain to the second complementary domain to produce a single-stranded gRNA.

The linker domain may be connected to the first complementary domain and the second complementary domain by a covalent or non-covalent bond.

The linker domain may connect the first complementary domain to the second complementary domain by a covalent or non-covalent bond.

The linker domain may be a sequence of 1-30 bases. The linker domain may comprise a sequence of 1-30 bases.

In an illustrative embodiment, the linker domain may be a sequence of 1-5, 5-10, 10-15, 15-20, 20-25, or 25-30 bases.

In an illustrative embodiment, the linker domain may comprise a sequence of 1-5, 5-10, 10-15, 15-20, 20-25, or 25-30 bases.

The linker domain is suitable for use in a single-stranded gRNA molecule and can be used to produce a single-stranded gRNA by connecting to the first strand and the second strand of a double-stranded gRNA or by connecting the first strand to the second strand by a covalent or non-covalent bond. The linker domain can be used to produce a single-stranded gRNA, by connecting a crRNA and a tracrRNA to a double-stranded gRNA, or by connecting a crRNA to a tracrRNA by a covalent or non-covalent bond.

The linker domain may have homology to or be derived from a natural sequence, such as a partial tracrRNA sequence.

In particular, some or all of the base sequences of the linker domain may have a chemical modification. The chemical modification may be methylation, acetylation, phosphorylation, phosphorothioate linkage, locked nucleic acid (LNA), 2'-Omethyl-3'-phosphorothioate (MS) or 2'-O-methyl-3'-thioPACE (MSP), with the present invention is not limited to them.

iv) Second complementary domain.

The second complementary domain includes a nucleic acid sequence complementary to the first complementary domain and has a sufficient degree of complementarity to form a double strand with the first complementary domain. The second complementary domain may include a base sequence complementary to the first complementary domain and a base sequence that is not complementary to the first complementary domain, such as a base sequence that does not form a double strand with the first complementary domain and may have a longer base sequence than the first complementary domain. domain.

In this case, the second complementary domain may be a sequence of 5-35 bases. The first complementary domain may include a sequence of 5-35 bases.

In an illustrative embodiment, the second complementary domain may be the sequence of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 bases.

In an illustrative embodiment, the second complementary domain may include the sequence of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 bases.

In addition, the second complementary domain may have homology to a naturally occurring second complementary domain or may be derived from a naturally occurring second complementary domain. In addition, the second complementary domain may have a difference in the base sequence of the second complementary domain according to naturally occurring species, may be derived from a second complementary domain found in a naturally occurring species, or may have partial or complete homology to the second complementary domain. domain contained in species existing in nature.

In an illustrative embodiment, the second complementary domain may have partial, i.e., at least 50% or greater or complete homology with the second complementary domain of Streptococcus pyogenes, Campylobacter jejuni, Streptococcus thermophilus, Staphylococcus aureus or Neisseria meningitides or with a second complementary domain derived therefrom .

For example, if the second complementary domain is the second complementary domain of Streptococcus pyogenes or a second complementary domain derived therefrom, then the second complementary domain may be 5'-UAGCAAGUUAAAAU-3 (SEQ ID NO: 44), or a base sequence characterized by partial, that is, at least 50% or greater homology with $-UAGCAAGUUAAAAU-3 (SEQ ID NO: 44), (the base sequence forming a double strand with the first complementary domain is underlined). In this case, the second complementary domain may further include (X) _n and/or (X) _m , resulting in

- 24 046169

5'-(X) _n UAGCAAGUUAAAAU(X) _m -3' (SEQ ID NO: 44).

X may be selected from the group consisting of bases A, T, U, and G, and each of n and m may be a number of bases, wherein n may be an integer from 1 to 15, and m may be an integer from 1 to 6. In this case, (X) _n may have n repeats of the same base or a combination of n bases A, T, U and G. Additionally, (X) _m may be m repeats of the same base or a mixture of m bases A, T, U and G.

As another example, if the second complementary domain is a Campylobacter jejuni second complementary domain or a second complementary domain derived therefrom, then the second complementary domain may be

-AAGAAAUUUAAAAAGGGACUAAAAU-3 (SEQ ID NO: 45), or a base sequence having partial, i.e., at least 50% or greater homology with 5'-AAGAAAUUUAAAAGGGACUAAAAU-3'(SEQ ID NO: 45), (the base sequence is underlined forming a double strand with the first complementary domain). In this case, the second complementary domain may further include (X) _n and/or (X) _m , resulting in 5'- (X)nAAAGAAAUUUAAAAAGGGACUAAAAU(X) _m -3' (SEQ III NO: 45).

X may be selected from the group consisting of bases A, T, U, and G, and each of n and m may be a number of bases, wherein n may be an integer from 1 to 15, and m may be an integer from 1 to 6. In this case, (X) _n may have n repeats of the same base or a combination of n bases A, T, U and G. Additionally, (X) _m may be m repeats of the same base or a combination of m bases A, T, U and G.

In another embodiment, the first complementary domain may have partial, i.e., at least 50% or greater or complete homology with the first complementary domain

For example, if the second complementary domain is the second complementary domain of Parcubacteria bacterium or a second complementary domain derived therefrom, then the second complementary domain may be

Parcubacteria bacterium (GWC2011_GWC2_44_17), Lachnospiraceae bacterium (MC2017), Butyrivibrio proteoclasiicus, Peregrinibacteria bacterium (GW2011_GWA_33_10), Acidaminococcus sp.

(BV3L6), Porphyromonas macacae, Lachnospiraceae bacterium (ND2006), Porphyromonas crevioricanis, Prevotella disiens, Moraxella bovoculi (237), Smiihella sp. (SC_KO8D17), Leptospira inadai. Lachnospiraceae bacterium (MA2020), Francisella novicida (UI 12), Candidatus Methanoplasma termitum or Eubacterium eligens, or the first complementary domain derived from them.

For example, if the second complementary domain is the second complementary domain of Parcubacteria bacterium or a second complementary domain derived therefrom, then the second complementary domain may be 5 -AAAUUUCUACU-3' (SEQ ID NO: 46), or a base sequence characterized by partial, that is, at least 50% or greater homology with 5'-AAAUUUCUACU-3' (SEQ ID NO: 46) (the base sequence forming a double strand with the first complementary domain is underlined). In this case, the second complementary domain may further include (X)n and/or (X)m, resulting in

5'-(X)nAAAUUUCUACU(X) _m -3' (SEQ ID NO: 46).

X may be selected from the group consisting of bases A, T, U, and G, and each of n and m may be a number of bases, wherein n may be an integer from 1 to 10, and m may be an integer from 1 to 6. In this case, (X) _n may have n repeats of the same base or a combination of n bases A, T, U and G. Additionally, (X) _m may be m repeats of the same base or a mixture of m bases A, T, U and G.

In particular, some or all of the base sequences of the second complementary domain may have a chemical modification. The chemical modification may be methylation, acetylation, phosphorylation, phosphorothioate linkage, locked nucleic acid (LNA), 2'-O-methyl-3'-phosphorothioate (MS) or 2'-O-methyl-3'-thioPACE (MSP) , but the present invention is not limited to them.

v) Proximal domain.

The proximal domain is a sequence of 1-20 bases located adjacent to the second complementary domain, and the domain is located 3'-terminal to the second complementary domain. In this case, the proximal domain can be used to form a double strand between its complementary base sequences.

In one illustrative embodiment, the proximal domain may represent

- 25 046169 fight a sequence of 5, 6, 7, 8, 8, 9, 10, 11, 12, 13, 14 or 15 bases.

In another embodiment, the proximal domain may include a sequence of 5, 6, 7, 8, 8, 9, 10, 11, 12, 13, 14, or 15 bases.

In addition, the proximal domain may have homology to a natural proximal domain or may be derived from a natural proximal domain. In addition, the proximal domain may have a difference in base sequence according to species existing in nature, may be derived from a proximal domain contained in a species existing in nature, or may have partial or complete homology with a proximal domain contained in a species existing in nature. existing in nature.

In an illustrative embodiment, the proximal domain may have partial, i.e., at least 50% or greater or complete homology with the proximal domain of Streptococcus pyogenes, Campylobacter jejuni, Streptococcus thermophilus, Staphylococcus aureus or Neisseria meningitides, or a proximal domain derived from them.

For example, if the proximal domain is the proximal domain of Streptococcus pyogenes or a proximal domain derived therefrom, then the proximal domain may be 5'-AAGGCUAGUCCG-3' (SEQ ID NO: 47) or a base sequence characterized by a partial, that is, at least 50% or greater homology with 5'-AAGGCUAGUCCG-3' (SEQ ID NO: 47).

In this case, the proximal domain may further include (X)n, resulting in 5'-AAGGCUAGUCCG(X) _n -3' (SEQ ID NO: 47).

X may be selected from the group consisting of bases A, T, U and G, and n may be a number of bases that is an integer from 1 to 15. In this case, (X) _n may have n repetitions of the same bases or a combination of n bases A, T, U and G.

As another example, if the proximal domain is a Campylobacter jejuni proximal domain or a proximal domain derived therefrom, the proximal domain may be 5'-AAAGAGUUUGC-3' (SEQ ID NO:48) or a base sequence characterized by at least 50 % or greater homology with ⁵ -AAAGAGUUUGC-3 (SEQ ID NO: 48).

In this case, the proximal domain may further include (X) _n , resulting in 5'-AAAGAGUUUGC(X) _n -3' (SEQ ID NO: 48).

X may be selected from the group consisting of bases A, T, U, and G, and n may be a number of bases that is an integer from 1 to 40. In this case, (X)n may have n repeats of the same bases or combination of n bases A, T, U and G.

In particular, some or all of the base sequences of the proximal domain may have a chemical modification. The chemical modification may be methylation, acetylation, phosphorylation, phosphorothioate linkage, locked nucleic acid (LNA), 2'-Omethyl-3'-phosphorothioate (MS) or 2'-O-methyl-3'-thioPACE (MSP), with the present invention is not limited to them.

vi) Tail domain.

A tail domain is a domain that can be selectively added to the 3' end of a single-stranded gRNA or a double-stranded gRNA. The tail domain may be a sequence of 1-50 bases or include a sequence of 1-50 bases. In this case, the tail domain can be used to form a double strand between its complementary base sequences.

In one illustrative embodiment, the tail domain may be a sequence of 1-5, 5-10, 10-15, 15-20, 20-25, 25-30, 30-35, 35-40, 40-45, or 45-50 grounds.

In an illustrative embodiment, the tail domain may comprise a sequence of 1-5, 5-10, 10-15, 15-20, 20-25, 25-30, 30-35, 35-40, 40-45, or 45-50 bases .

In addition, the tail domain may have homology to the natural tail domain or may be derived from the natural tail domain. In addition, the tail domain may have a difference in base sequence according to species existing in nature, may be derived from a tail domain contained in a species existing in nature, or may have partial or complete homology with a tail domain contained in a species existing in nature. existing in nature.

In one illustrative embodiment, the tail domain may have partial, i.e., at least 50% or greater or complete homology with the tail domain of Streptococcus pyogenes, Campylobacter jejuni, Streptococcus thermophilus, Staphylococcus aureus or Neisseria meningitides or a tail domain derived from them.

For example, if the tail domain is the tail domain of Streptococcus pyogenes or a tail domain derived therefrom, the tail domain may be 5'-UUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC-3' (SEQ ID NO: 49) or a base sequence characterized by a partial, that is, at least 50% or greater homology with

- 26 046169

5'-UUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC-3' (SEQ ID NO: 49).

In this case, the tail domain may further include (X) _n , resulting in 5'-UUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC(X) _n -3' (SEQ ID NO: 49).

X may be selected from the group consisting of bases A, T, U and G, and n may be a number of bases that is an integer from 1 to 15. In this case, (X) _n may have n repetitions of the same bases or a combination of n bases such as A, T, U and G.

As another example, if the tail domain is the tail domain of Campylobacter jejuni or a tail domain derived therefrom, the tail domain may be 5'-GGGACUCUGCGGGGUUACAAUCCCCUAAAACCGCUUUU-3' (SEQ ID NO: 50) or a base sequence characterized by partial, i.e. at least 50% or greater homology with 5'_GGGACUCUGCGGGGGUUACAAUCCCCUAAAACCGCUUUU-3' (SEQ ID NO: 50).

In this case, the tail domain may further include (X) _n , resulting in 5'-GGGACUCUGCGGGGGUUACAAUCCCCUAAAACCGCUUUU(X) _n -3' (SEQ ID NO: 50).

X may be selected from the group consisting of bases A, T, U and G, and n may be a number of bases that is an integer from 1 to 15. In this case, (X) _n may have n repetitions of the same bases or combination of n bases A, T, U and G.

In another embodiment, the tail domain may include a sequence of 1-10 bases at the 3' end involved in in vitro or in vivo transcription.

For example, when using the T7 promoter in in vitro transcription of a gRNA, the tail domain can be an arbitrary sequence of bases present at the 3' end of the DNA template. In addition, when using the U6 promoter in in vivo transcription, the tail domain may be UUUUUU, when using the H1 promoter in transcription, the tail domain may be UUUU, and when using the pol-III promoter, the tail domain may include several uracil bases or alternative bases.

In particular, some or all of the tail domain base sequences may have a chemical modification. The chemical modification may be methylation, acetylation, phosphorylation, phosphorothioate linkage, locked nucleic acid (LNA), 2'-Omethyl-3'-phosphorothioate (MS) or 2'-O-methyl-3'-thioPACE (MSP), with the present invention is not limited to them.

The gRNA may include multiple domains as described above, and therefore the length of the nucleic acid sequence can be adjusted according to the domain contained in the gRNA, and interactions may occur within the strands, in the three-dimensional structure or active form of the gRNA, or between these strands due to each domain.

gRNA can be called a single-stranded gRNA (a single RNA molecule) or a double-stranded gRNA (comprising more than one, usually two separate RNA molecules).

Double-stranded gRNA.

A double-stranded gRNA consists of a first strand and a second strand.

In this case, the first strand may consist of 5'-[targeting domain]-[first complementary domain]-3', and the second strand may consist of 5'-[second complementary domain][proximal domain]-3' or 5'- [second complementary domain]-[proximal domain][tail domain]-In this case, the first strand can be called crRNA and the second strand can be called tracrRNA.

First thread.

Guiding domain.

In the first strand, the targeting domain includes a complementary targeting sequence that can form a complementary linkage to a target sequence in the target gene or nucleic acid. A guide sequence is a nucleic acid sequence complementary to a target sequence in a target gene or nucleic acid that is, for example, at least 70%, 75%, 80%, 85%, 90% or 95%, or greater complementarity or complete complementarity . The targeting domain is thought to allow the gRNA-Cas complex, that is, the CRISPR complex, to specifically interact with a target gene or nucleic acid.

In this case, the targeting domain may be a sequence of 5-50 bases or include a sequence of 5-50 bases. For example, the targeting domain may be or include a sequence of 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 bases.

In addition, the targeting domain may include a targeting sequence.

In this case, the targeting sequence may be a complementary base sequence that can form a complementary relationship with a target sequence in the target gene or nucleic acid that is, for example, at least 70%, 75%, 80%, 85%, 90%, or 95% or greater complementary or fully complementary

- 27 046169 noness.

In an illustrative embodiment, the targeting sequence may be a nucleic acid sequence complementary to a target gene, that is, a target sequence of an unsaturated fatty acid biosynthesis-associated factor such as a FAD gene, preferably a FAD2 gene, a FAD3 gene, a FAD6 gene, a FAD7 gene, or a FAD8 gene, which is characterized, for example, by at least 70%, 75%, 80%, 85%, 90% or 95% or greater complementarity or complete complementarity.

In this case, the guide sequence may be a sequence of 5-50 bases or include a sequence of 5-50 bases. For example, the guide sequence may be or include a sequence of 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 bases.

In one illustrative embodiment, the targeting sequence is a nucleic acid sequence complementary to the target sequence of the FAD2 gene. The guide sequence may be or include a sequence of 5-50 bases. For example, the guide sequence may be or include a sequence of 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 bases.

In one illustrative embodiment, the guide sequence is a nucleic acid sequence complementary to the target sequence of the FAD3 gene. The guide sequence may be or include a sequence of 5-50 bases. For example, the guide sequence may be or include a sequence of 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 bases.

In one illustrative embodiment, the guide sequence is a nucleic acid sequence complementary to the target sequence of the FAD6 gene. The guide sequence may be or include a sequence of 5-50 bases. For example, the guide sequence may be or include a sequence of 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 bases.

In one illustrative embodiment, the guide sequence is a nucleic acid sequence complementary to the target sequence of the FAD7 gene. The guide sequence may be or include a sequence of 5-50 bases. For example, the guide sequence may be or include a sequence of 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 bases.

In one illustrative embodiment, the guide sequence is a nucleic acid sequence complementary to the target sequence of the FAD8 gene. The guide sequence may be or include a sequence of 5-50 bases. For example, the guide sequence may be or include a sequence of 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 bases.

In this case, the guide sequences, target sequences of the target gene, that is, factors associated with the biosynthesis of unsaturated fatty acids, such as the FAD2 gene, are given above in table. 1, but the present invention is not limited to them.

In particular, the targeting domain may include a targeting sequence and an additional base sequence.

In this case, the additional base sequence may be a sequence of 1-35 bases. For example, the additional base sequence may be a sequence of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 bases.

In one illustrative embodiment, the additional base sequence may include one guanine (G) base or two GG bases.

In this case, the additional base sequence may be located at the 5' end of the targeting domain or at the 5' end of the targeting sequence.

The additional base sequence may be located at the 3' end of the targeting domain or at the 3' end of the targeting sequence.

First complementary domain.

The first complementary domain includes a nucleic acid sequence complementary to the second complementary domain of the second strand and is a domain having sufficient complementarity to form a double strand with the second complementary domain.

In this case, the first complementary domain may be or include a sequence of 5-35 bases. For example, the first complementary domain may be or include the sequence of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 bases.

The first complementary domain may have homology to a naturally occurring first complementary domain or may be derived from a naturally occurring first complementary domain. In addition, the first complementary domain may differ in base sequence in

- 28 046169 according to a species existing in nature, may be derived from a first complementary domain contained in a species existing in nature, or may be characterized by partial or complete homology with a first complementary domain contained in a species existing in nature.

In one illustrative embodiment, the first complementary domain may have partial, i.e., at least 50% or greater or complete homology with the first complementary domain of Streptococcus pyogenes, Campylobacter jejum, Streptococcus thermophilus, Staphylococcus aureis, or Νβύ8βηα meningitides, or with the first complementary domain derived out of him.

In particular, the first complementary domain may include an additional base sequence that does not undergo complementary binding to the second complementary domain of the second strand.

In this case, the additional base sequence may be a sequence of 1-15 bases. For example, the additional base sequence may be a sequence of 1-5, 5-10, or 10-15 bases.

In particular, part or all of the base sequences of the targeting domain and/or the first complementary domain may have a chemical modification. The chemical modification may be methylation, acetylation, phosphorylation, phosphorothioate linkage, locked nucleic acid (LNA), 2'-O-methyl-3'-phosphorothioate (MS) or 2'-O-methyl-3'-thioPACE (MSP) , but the present invention is not limited to them.

Therefore, the first strand may consist of 5'-[targeting domain]-[first complementary domain]-3', as described above.

In addition, the first strand may optionally include additional base sequence.

In one example, the first thread may be

-(N _MinneHb )-(Q) _m -3 or ' -(X) _a -(N _MI nn _e Hb)-(X)b-(Q)m-(X)c-3 '.

Here, a _target is a base sequence capable of forming a complementary bond with a target sequence in a target gene or nucleic acid, and a region of base sequence that can be changed according to the target sequence in the target gene or nucleic acid.

In one illustrative embodiment, the Hm _target may be a base sequence capable of forming a complementary linkage to a target gene, that is, the target sequence of an unsaturated fatty acid biosynthesis-associated factor, such as a FAD gene, preferably a FAD2 gene, a FAD3 gene, a FAD6 gene, a FAD7 gene or the FAD8 gene.

Here, (Q) _m is a sequence of bases comprising a first complementary domain that can form a complementary bond with a second complementary domain of the second strand. (Q) _m may be a sequence having partial or complete homology with the first complementary domain of a species existing in nature, and the base sequence of the first complementary domain may be changed according to the species of origin. Each Q may be independently selected from the group consisting of A, U, C and G, and m may be a number of bases that is an integer from 5 to 35.

For example, if the first complementary domain has partial or complete homology to the first complementary domain of Streptococcus pyogenes or a Streptococcus pyogenes-derived first complementary domain, then (Q) _m may be 5'-GUUUUAGAGCUA-3' (SEQ ID NO: 42) or the sequence bases having at least 50% or greater homology with $-GUUUUAGAGCUA-3 (SEQ ID NO. 42).

As another example, if the first complementary domain has partial or complete homology with the first complementary domain of Campylobacter jejuni or a Campylobacter jejuni-derived first complementary domain, then (Q) _m may be 5 -GUUUUAGUCCCUUUUUAAAUUUCUU-3 (SEQ ID NO: 43) or a base sequence having at least 50% or greater homology with 5'-GUUUUAGUCCCUUUUUAAAUUUCUU-3' (SEQ ID NO: 43).

As another example, if the first complementary domain has partial or complete homology to the first complementary domain of Streptococcus thermophilus or a Streptococcus thermophilus-derived first complementary domain, then (Q) _m may be 5'-GUUUUAGAGCUGUGUUGUUUCG-3' (SEQ ID NO: 51 ) or a base sequence having at least 50% or greater homology with

- 29 046169

5'-GUUUUAGAGCUGUGUUGUUUCG-3' (SEQ ID NO: 51).

In addition, each of (X) _a , (X)b and (X) _c selectively represents an additional sequence of bases, where each X can be independently selected from the group consisting of A, U, C and G, and each of a , b and c can represent the number of bases, which is 0 or an integer from 1 to 20.

Second thread.

The second strand may consist of a second complementary domain, a proximal domain, and may selectively include a tail domain.

Second complementary domain.

In the second strand, the second complementary domain includes a nucleic acid sequence complementary to the first complementary domain and has a sufficient degree of complementarity to form a double strand with the first complementary domain. The second complementary domain may include a base sequence complementary to the first complementary domain and a base sequence that is not complementary to the first complementary domain, such as a base sequence that does not form a double strand with the first complementary domain and may have a longer base sequence than the first complementary domain. domain.

In this case, the second complementary domain may be a sequence of 5-35 bases or include a sequence of 5-35 bases. For example, the second complementary domain may be or include the sequence of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 bases, but the present invention is not limited to them.

The second complementary domain may have homology to a naturally occurring second complementary domain or may be derived from a naturally occurring second complementary domain. In addition, the second complementary domain may have a difference in its base sequence in accordance with species existing in nature, may be derived from a second complementary domain contained in species existing in nature, or may have partial or complete homology with the second complementary domain, contained in species existing in nature.

In one illustrative embodiment, the second complementary domain may have partial, i.e., at least 50% or greater or complete homology with the second complementary domain of Streptococcus pyogenes, Campylobacter jejuni, Streptococcus thermophilus, Staphylococcus aureus or Neisseria meningitides or with a second complementary domain derived from them.

In particular, the second complementary domain may further include an additional base sequence that does not undergo complementary binding to the first complementary domain of the first strand.

In this case, the additional base sequence may be a sequence of 1-25 bases. For example, the additional base sequence may be a sequence of 1-5, 5-10, 10-15, 15-20, or 20-25 bases.

Proximal domain.

In the second strand, the proximal domain is a sequence of 1-20 bases, and a domain located in the direction relative to the 3' end of the second complementary domain. For example, the proximal domain may be or include a sequence of 5, 6, 7, 8, 8, 9, 10, 11, 12, 13, 14, or 15 bases.

In this case, the proximal domain may have a double-stranded connection between its complementary base sequences.

In addition, the proximal domain may have homology to a natural proximal domain or may be derived from a natural proximal domain. In addition, the proximal domain may have a difference in base sequence according to species existing in nature, may be derived from the proximal domain of a species existing in nature, or may have partial or complete homology with the proximal domain of a species existing in nature.

In one illustrative embodiment, the proximal domain may have partial, i.e., at least 50% or greater or complete homology with the proximal domain of Streptococcus pyogenes, Campylobacter jejuni, Streptococcus thermophilus, Staphylococcus aureus or Neisseria meningitides, or a proximal domain derived from them.

Tail domain.

Specifically, in the second strand, the tail domain may be a domain selectively added to the 3' end of the second strand, and the tail domain may be or include a sequence of 1 to 50 bases. For example, the tail domain may be or include the sequence of 1-5, 5-10, 10-15, 15-20, 20-25, 25-30, 30-35, 35-40, 40-45, or 45-50 grounds.

- 30 046169

In this case, the tail domain may have a double-stranded relationship between its complementary base sequences.

In addition, the tail domain may have homology to the natural tail domain or may be derived from the natural tail domain. In addition, the tail domain may have a difference in base sequence according to species existing in nature, may be derived from a tail domain contained in a species existing in nature, or may have partial or complete homology with a tail domain contained in a species existing in nature. existing in nature.

In one illustrative embodiment, the tail domain may have partial, i.e., at least 50% or greater or complete homology with the tail domain of Streptococcus pyogenes, Campylobacter jejuni, Streptococcus thermophilus, Staphylococcus aureus or Neisseria meningitides or a tail domain derived from them.

In another embodiment, the tail domain may include a sequence of 1-10 bases at the 3' end involved in in vitro or in vivo transcription.

For example, when using the T7 promoter in in vitro transcription of a gRNA, the tail domain can be an arbitrary sequence of bases present at the 3' end of the DNA template. In addition, when using the U6 promoter in in vivo transcription, the tail domain may be UUUUUU, when using the H1 promoter in transcription, the tail domain may be UUUU, and when using the pol-III promoter, the tail domain may include several uracil bases or alternative bases.

In particular, part or all of each base sequence of the second complementary domain, proximal domain and/or tail domain may have a chemical modification. The chemical modification may be methylation, acetylation, phosphorylation, phosphorothioate linkage, locked nucleic acid (LNA), 2'-O-methyl-3'-phosphorothioate (MS) or 2'-O-methyl-3'-thioPACE (MSP) , but the present invention is not limited to them.

Therefore, the second strand may consist of 5'-[second complementary domain][proximal domain]-3' or 5'-[second complementary domain]-[proximal domain][tail domain]-3', as described above.

In addition, the second strand may selectively include additional base sequence.

In one illustrative embodiment, the second strand may be 5'-(Z) _h (P)k-3' or 5'-(X)d-(Z) _h- (X)e-(P)k-(X ) _r 3'.

In another embodiment, the second strand may be 5'-(Z)h-(P) _k- (F)i-3' or 5'(X)d-(Z)h-(X)e-(P) k-(X)f-(F) ₁ -3'.

Here, (Z) _h is a sequence of bases comprising a second complementary domain that can form a complementary bond with the first complementary domain of the first strand. (Z) _h may be a sequence having partial or complete homology with the second complementary domain of a species existing in nature, and the base sequence of the second complementary domain may be modified according to the species of origin. Each Z may be independently selected from the group consisting of A, U, C, and G, and h may be a number of bases that is an integer from 5 to 50.

For example, if the second complementary domain has partial or complete homology to the second complementary domain of Streptococcus pyogenes or a second complementary domain derived therefrom, then (Z) _h may be 5'-UAGCAAGUUAAAAU-3' (SEQ ID NO: 44) or the sequence bases having at least 50% or greater homology with 5'-UAGCAAGUUAAAAU-3' (SEQ ID NO: 44).

As another example, if the second complementary domain has partial or complete homology to the second complementary domain of Campylobacter jejuni or a second complementary domain derived therefrom, then (Z)h may be 5 -AAGAAAUUUAAAAAGGGACUAAAAU-3 (SEQ ID NO: 45) or a base sequence having at least 50% or greater homology with 5'-AAGAAAUUUAAAAAGGGACUAAAAU-3' (SEQ III NO: 45).

In yet another example, if the second complementary domain has partial or complete homology to the second complementary domain of Streptococcus thermophilus or a second complementary domain derived therefrom, then (Z)h may be 5'-CGAAACAACACAGCGAGUUAAAAU-3' (SEQ ID NO: 52 ) or a base sequence having at least 50% or greater homology with 5'-CGAAACAACACAGCGAGUUAAAAU-3' (SEQ ID NO: 52).

(P) _k is a sequence of bases comprising a proximal domain, which may be characterized by partial or complete homology with the proximal domain of the species derived from it, then

5'-AAGGCUUAGUCCG-3' (SEQ Ш NO:

at least

5'-AAGGCUUAGUCCG-3' (SEQ IE) NO: 53).

existing in nature, and the base sequence of the proximal domain can be modified according to the species of origin. Each P may be independently selected from the group consisting of A, U, C, and G, and k may be a number of bases that is an integer from 1 to 20.

For example, if the proximal domain has partial or complete homology to the proximal domain of Streptococcus pyogenes or a proximal domain derived therefrom, then (P) _k may be 5'-AAGGCUAGUCCG-3' (SEQ ID NO: 47) or a base sequence characterized by at least 50% or greater homology with 5'-AAGGCUAGUCCG-3' (SEQ ID NO: 47).

As another example, if the proximal domain has partial or complete homology to the Campylobacter jejuni proximal domain or a proximal domain derived therefrom, then (P) _k may be 5'-AAAGAGUUUGC-3' (SEQ ID NO: 48) or the sequence bases having at least 50% or greater homology with 5'-AAAGAGUUUGC-3' (SEQ III NO: 48).

As a further example, if the proximal domain has partial or complete homology with the proximal domain of Streptococcus thermophilus or the proximal domain, (P) _k may be 53) or a base sequence having 50% or greater homology with (F)i may be the sequence bases comprising a tail domain and having partial or complete homology with the tail domain of species existing in nature, and the base sequence of the tail domain may be modified according to the species of origin. Each F may be independently selected from the group consisting of A, U, C and G, ai may be a number of bases that is an integer from 1 to 50.

For example, if the tail domain is characterized by partial or complete homology to the tail domain of Streptococcus pyogenes or a tail domain derived therefrom, then (F)i may be 5'-UUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC-3' (SEQ ID NO: 49) or a base sequence characterized by at least 50% or greater homology with 5'-UUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC-3' (SEQ ID NO: 49).

As another example, if the tail domain has partial or complete homology to the tail domain of Campylobacter jejuni or a tail domain derived therefrom, then (F) may be

5'-GGGACUCUGCGGGGGUUACAAUCCCCUAAAACCGCUUUU-3'(SEQIDNO: 50) or a base sequence having at least 50% or greater homology with 5'-GGGACUCUGCGGGGUUACAAUCCCCCUAAAACCGCUUUU-3' (SEQ III NO: 50).

As a further example, if the tail domain has partial or complete homology with the tail domain of Streptococcus thermophilus or a tail domain derived therefrom, then (F)i may be

5'-UACUCAACUUGAAAAGGUGGCACCGAUUCGGUGUUUUU-3' (SEQ ID NO: 54) _is a base sequence having at least 50% or greater homology with 5'-UACUCAACUUGAAAAGGUGGCACCGAUUCGGUGUUUUU-3' (SEQ ID NO: 54).

In addition, (F)i may include a sequence of 1-10 bases at the 3'-terminus involved in in vitro or in vivo transcription.

For example, when using the T7 promoter for in vitro transcription of gRNA, the tail domain can be an arbitrary sequence of bases present at the 3' end of the DNA template. In addition, when using the U6 promoter in in vivo transcription, the tail domain may be UUUUUU, when using the H1 promoter in transcription, the tail domain may be UUUU, and when using the pol-III promoter, the tail domain may include several uracil bases or alternative bases.

Additionally, (X)d, (X) _e , and (X)f may be selectively added base sequences, where each X may be independently selected from the group consisting of A, U, C, and G, and each d, e and f can represent the number of bases, which is 0 or an integer from 1 to 20.

Single-stranded gRNA.

Single-stranded gRNA can be classified into two types.

i) Single-stranded gRNA.

First, there is a single-stranded gRNA in which the first strand or the second strand of a double-stranded gRNA is linked by a linker domain, and in this case, a single-stranded gRNA consists of 5'[first strand]-[linker domain]-[second strand]-3' .

In particular, a single-stranded gRNA may consist of a 5'-[targeting domain]-[first complex

- 32 046169 mental domain]-[linker domain]-[second complementary domain]-[proximal domain]-3' or 5'-[targeting domain]-[first complementary domain]-[linker domain][second complementary domain]- [proximal domain]-[tail domain]-3'.

Each domain, except the linker domain, is similar to the description of each domain of the first and second strands of a double-stranded gRNA.

Linker domain.

In a single-stranded gRNA, a linker domain is a domain connecting a first strand and a second strand, and in particular is a nucleic acid sequence that connects a first complementary domain to a second complementary domain to form a single-stranded gRNA. Here, the linker domain may be connected to the first complementary domain and the second complementary domain by a covalent bond or a non-covalent bond, or may connect the first complementary domain to the second complementary domain by a covalent or non-covalent bond.

The linker domain may be or include a sequence of 1-30 bases. For example, the linker domain may be or include a sequence of 1-5, 5-10, 10-15, 15-20, 20-25 or 25-30 bases.

The linker domain is suitable for use in a single-stranded gRNA molecule and can be connected to the first strand and the second strand of a double-stranded gRNA or can connect the first strand to the second strand by a covalent or non-covalent bond for use in producing a single-stranded gRNA. The linker domain can be connected to crRNA and tracrRNA by double-stranded gRNA or can connect crRNA to tracrRNA by a covalent or non-covalent linkage for use in producing single-stranded gRNA.

The linker domain may have homology to or be derived from a natural sequence, such as a partial tracrRNA sequence.

In particular, some or all of the base sequences of the linker domain may have a chemical modification. The chemical modification may be methylation, acetylation, phosphorylation, phosphorothioate linkage, locked nucleic acid (LNA), 2'-Omethyl-3'-phosphorothioate (MS) or 2'-O-methyl-3'-thioPACE (MSP), with the present invention is not limited to them.

Therefore, a single-stranded gRNA may consist of 5'-[targeting domain]-[first complementary domain]-[linker domain]-[second complementary domain]-[proximal domain]-3' or 5'-[targeting domain]-[first complementary domain]-[linker domain][second complementary domain]-[proximal domain]-[tail domain]-3', as described above.

In addition, a single-stranded gRNA may selectively include additional base sequence.

In one illustrative embodiment, the single-stranded gRNA may be '-(N _MinneHb )-(Q) _m- (L)j-(Z) _h- (P) _k -3' or

5'-(Ntarget)-(Q) _m -(L) _j -(Z) _h -(P) _k -(F) ₁ -3'.

In another embodiment, the single-stranded gRNA may be

I5'-(X) _a -(N _MmneH b)-(X)b-(Q)m-(X)c-(L) _j -(X) _d -(Z) ₁₁ -(X) _e -( P) _k -(X)r3'^H

5'-(X) _a -(N _MinneHb )-(X) _b -(Q) _m -(X) _c -(L)j-(X) _d -(Z)h-(X) _e -(P ) _k -(X)r(F)i-3'.

Here, a _target is a base sequence capable of forming a complementary bond with a target sequence in the target gene or nucleic acid, and a region of the base sequence capable of varying according to the target sequence in the target gene or nucleic acid.

In one illustrative embodiment, _{the target} is a base sequence capable of forming a complementary bond to a target gene, that is, the target sequence of an unsaturated fatty acid biosynthesis-associated factor, such as a FAD gene, preferably a FAD2 gene, a FAD3 gene, a FAD6 gene, a FAD7 gene, or FAD8 gene.

(Q) _m includes a sequence of bases including a first complementary domain that can form a complementary bond with a second complementary domain. (Q) _m may be a sequence having partial or complete homology with the first complementary domain of a species existing in nature, and the base sequence of the first complementary domain may be changed according to the species of origin. Each Q may be independently selected from the group consisting of A, U, C, and G, and m may be a number of bases that is an integer from 5 to 35.

For example, if the first complementary domain is characterized by partial or complete homology

- 33 046169 her with the first complementary domain of Streptococcus pyogenes or the first complementary domain derived therefrom, then (Q)m may be 5'-GUUUUAGAGCUA-3' (SEQ ID NO: 42) or a base sequence characterized by at least 50 % or greater homology with 5'-GUUUUAGAGCUA-3' (SEQ III NO: 42).

As another example, if the first complementary domain has partial or complete homology to the first complementary domain of Campylobacter jejuni or a first complementary domain derived therefrom, then (Q) _m may be 5'-GUUUUAGUCCCUUUUUAAAUUUCUU-3' (SEQ ID NO: 43 ), or a base sequence having at least 50% or greater homology with 5-GUUUUAGUCCCUUUUUAAAUUUCUU-3' (SEQ ID NO: 43).

As a further example, if the first complementary domain has partial or complete homology with the first complementary domain of Streptococcus thermophilus or a first complementary domain derived therefrom, then (Q) _m may be 5'-GUUUUAGAGCUGUGUUGUUUCG-3' (SEQ ID NO: 51 ) or a base sequence having at least 50% or greater homology with 5'-GUUUUAGAGCUGUGUUGUUUCG-3' (SEQ ID NO: 51).

In addition, (L)j is a base sequence including a linker domain and connecting the first complementary domain to the second complementary domain, thereby obtaining a single-stranded gRNA. Each L may be independently selected from the group consisting of A, U, C, and G, and j may be a number of bases that is an integer from 1 to 30.

(Z) _h is a sequence of bases comprising a second complementary domain that may have a complementary relationship with the first complementary domain. (Z) _h may be a sequence having partial or complete homology with the second complementary domain of species existing in nature, and the base sequence of the second complementary domain may be changed according to the species of origin. Each Z can be independently selected from the group consisting of A, U, C and G, and h is the number of bases, which can be an integer from 5 to 50.

For example, if the second complementary domain has partial or complete homology to the second complementary domain of Streptococcus pyogenes or a second complementary domain derived therefrom, then (Z) _h may be 5'-UAGCAAGUUAAAAU-3' (SEQ ID NO: 44) or the sequence bases having at least 50% or greater homology with 5'-UAGCAAGUUAAAAU-3' (SEQ ID NO: 44).

As another example, if the second complementary domain has partial or complete homology to the second complementary domain of Campylobacter jejuni or a second complementary domain derived therefrom, then (Z) _h may be 5'-AAGAAAUUUAAAAAGGGACUAAAAU-3' (SEQ ID NO: 45 ) or a base sequence having at least 50% or greater homology with 5'-AAGAAAUUUAAAAAGGGACUAAAAU-3' (SEQ ID NO:45).

In yet another example, if the second complementary domain has partial or complete homology to the second complementary domain of Streptococcus thermophilus or a second complementary domain derived therefrom, then (Z) _h may be 5'-CGAAACAACACAGCGAGUUAAAAU-3' (SEQ III NO: 52 ) or a base sequence having at least 50% or greater homology with 5'-CGAAACAACACAGCGAGUUAAAAU-3' (SEQ ID NO: 52).

(P)k is a base sequence including a proximal domain, which may have partial or complete homology with the proximal domain of species existing in nature, and the base sequence of the proximal domain may be modified according to the species of origin. Each P may be independently selected from the group consisting of A, U, C, and G, and k may be a number of bases that is an integer from 1 to 20.

For example, if the proximal domain is characterized by partial or complete homology with the proximal domain of Streptococcus pyogenes or a proximal domain derived therefrom, then (P) _k may be 5'-AAGGCUAGUCCG-3' (SEQ ID NO: 47) or a base sequence characterized by at least 50% or greater homology with 5'.AAGGCUAGUCCG-3' (SEQ ID NO: 47).

As another example, if the proximal domain has partial or complete homology to the Campylobacter jejuni proximal domain or a proximal domain derived therefrom, then (P) _k may be a 5'-AAAGAGUUUGC-3 (SEQ ID NO: 48) or base sequence , characterized by at least 50% or greater homology with 5'.AAAGAGUUUGC-3' (SEQ III NO: 48).

- 34 046169

As a further example, if the proximal domain has partial or complete homology to the proximal domain of Streptococcus thermophilus or a proximal domain derived therefrom, then (P) _k may be $-AAGGCUUAGUCCG-3 (SEQ ID NO: 53) or a base sequence having at least 50% or greater homology with 5'-AAGGCUUAGUCCG-3' (SEQ ID NO: 53).

(F)i may be a base sequence including a tail domain and having partial or complete homology with the tail domain of a species existing in nature, and the base sequence of the tail domain may be modified according to the species of origin. Each F may be independently selected from the group consisting of A, U, C and G, and i may be a number of bases that is an integer from 1 to 50.

For example, if the tail domain is characterized by partial or complete homology to the tail domain of Streptococcus pyogenes or a tail domain derived therefrom, then (F)i may be 5'-UUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC-3' (SEQ ID NO: 49) or a base sequence characterized by at least 50% or greater homology with 5'-UUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC-3' (SEQ ID NO: 49).

As another example, if the tail domain has partial or complete homology to the tail domain of Campylobacter jejuni or a tail domain derived therefrom, then (F)i may be

5'-GGGACUCUGCGGGGGUUACAAUCCCCUAAAACCGCUUUU-3' (SEQ ID NO: 50) or a base sequence having at least 50% or greater homology with 5'-GGGACUCUGCGGGGUUACAAUCCCCCUAAAACCGCUUUU-3' (SEQ ID NO: 50).

As a further example, if the tail domain has partial or complete homology to the tail domain of Streptococcus thermophilus or a tail domain derived therefrom, then (F)i may be _{5'_UACUCAACUUGAAAAGGUGGCACCGAUUCGGUGUUUUU} -3' (SEQ ID NO: 54) or the base sequence , characterized by at least 50% or greater homology with 5'-UACUCAACUUGAAAGGUGGCACCGAUUCGGUGUUUUU-3' (SEQ ID NO: 54).

In addition, (F)i may include a sequence of 1-10 bases at the 3' end involved in in vitro or in vivo transcription.

For example, when using the T7 promoter in in vitro transcription of a gRNA, the tail domain can be an arbitrary sequence of bases present at the 3' end of the DNA template. In addition, when using the U6 promoter in in vivo transcription, the tail domain may be UUUUUU, when using the H1 promoter in transcription, the tail domain may be UUUU, and when using the pol-III promoter, the tail domain may include several uracil bases or alternative bases.

Additionally, (X) _a , (X) _b , (X) _c , (X) _d , (X) _e , and (X)f may be selectively added base sequences, where each X may be independently selected from the group, consisting of A, U, C and G, and each of a, b, c, d, e and f can represent a number of bases that is 0 or an integer from 1 to 20.

ii) Single-stranded gRNA.

Secondly, the single-stranded gRNA may be a single-stranded gRNA consisting of a targeting domain, a first complementary domain and a second complementary domain, in which case the single-stranded gRNA may be composed of:

5'-[second complementary domain]-[first complementary domain]-[targeting domain]-3' or

5'-[second complementary domain]-[linker domain]-[first complementary domain][targeting domain]-3'.

Guiding domain.

In a single-stranded gRNA, the targeting domain includes a complementary targeting sequence capable of forming a complementary linkage with a target sequence in the target gene or nucleic acid. The guide sequence may be a nucleic acid sequence complementary to a target sequence in the target gene or nucleic acid that is, for example, at least 70%, 75%, 80%, 85%, 90% or 95% or greater complementarity or complete complementarity. The targeting domain is thought to allow the gRNA-Cas complex, that is, the CRISPR complex, to specifically interact with a target gene or nucleic acid.

In this case, the targeting domain may be or include a sequence of 5-50 bases. For example, the targeting domain may be or include a sequence of 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 bases.

In addition, the targeting domain may include a targeting sequence.

In this case, the guide sequence may be complementary

- 35 046169 a base sequence capable of forming a complementary relationship with a target sequence in a target gene or nucleic acid, which is characterized by, for example, at least 70%, 75%, 80%, 85%, 90% or 95% or greater complementarity or complete complementarity.

In one illustrative embodiment, the targeting sequence may be a nucleic acid sequence complementary to a target gene, that is, a target sequence of an unsaturated fatty acid biosynthesis-associated factor such as a FAD gene, preferably a FAD2 gene, a FAD3 gene, a FAD6 gene, a FAD7 gene, or a FAD8 gene which is characterized, for example, by at least 70%, 75%, 80%, 85%, 90% or 95% or greater complementarity or complete complementarity.

In this case, the guide sequence may be or include a sequence of 5-50 bases. For example, the guide sequence may be or include a sequence of 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 bases.

In one illustrative embodiment, the targeting sequence may be a nucleic acid sequence complementary to the target sequence of the FAD2 gene. The guide sequence may be or include a sequence of 5-50 bases. For example, the guide sequence may be or include a sequence of 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 bases.

In one illustrative embodiment, the targeting sequence may be a nucleic acid sequence complementary to the target sequence of the FAD3 gene. The guide sequence may be or include a sequence of 5-50 bases. For example, the guide sequence may be or include a sequence of 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 bases.

In one illustrative embodiment, the targeting sequence may be a nucleic acid sequence complementary to the target sequence of the FAD6 gene. The guide sequence may be or include a sequence of 5-50 bases. For example, the guide sequence may be or include a sequence of 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 bases.

In one illustrative embodiment, the targeting sequence may be a nucleic acid sequence complementary to the target sequence of the FAD7 gene. The guide sequence may be or include a sequence of 5-50 bases. For example, the guide sequence may be or include a sequence of 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 bases.

In one illustrative embodiment, the targeting sequence may be a nucleic acid sequence complementary to the target sequence of the FAD8 gene. The guide sequence may be or include a sequence of 5-50 bases. For example, the guide sequence may be or include a sequence of 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 bases.

In this case, the target sequences of the target genes, that is, the factor associated with the biosynthesis of unsaturated fatty acids, such as the FAD2 gene, for the guide sequence are given above in table. 1, but the present invention is not limited to them.

In particular, the targeting domain may include a targeting sequence and an additional base sequence.

In this case, the additional base sequence may be a sequence of 1-35 bases. For example, the additional base sequence may be a sequence of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 bases.

In one illustrative embodiment, the additional base sequence may be a single guanine (G) base sequence or a double GG base sequence.

In this case, the additional base sequence may be located at the 5' end of the targeting domain or at the 5' end of the targeting sequence.

The additional base sequence may be located at the 3' end of the targeting domain or at the 3' end of the targeting sequence.

First complementary domain.

The first complementary domain is a nucleic acid sequence comprising a nucleic acid sequence complementary to the second complementary domain and having a sufficient degree of complementarity to form a double strand with the second complementary domain.

In this case, the first complementary domain may be or include a sequence of 5-35 bases. For example, the first complementary domain may be or include the sequence of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 bases.

- 36 046169

The first complementary domain may have homology to a naturally occurring first complementary domain or may be derived from a naturally occurring first complementary domain. In addition, the first complementary domain may have a difference in the base sequence of the first complementary domain according to naturally occurring species, may be derived from a first complementary domain found in a naturally occurring species, or may have partial or complete homology to the first complementary domain. domain contained in species existing in nature.

In one illustrative embodiment, the first complementary domain may have partial, i.e., at least 50% or greater or complete homology with the first complementary domain

Parcubacteria bacterium (GWC2011_GWC2_44_17), Lachnospiraceae bacterium (MC2017), Butyrivibrio proteoclasiicus, Peregrinibacteria bacterium (GW2011_GWA_33_10), Acidaminococcus sp.

(BV3L6), Porphyromonas macacae, Lachnospiraceae bacterium (ND2006), Porphyromonas crevioricanis, Prevotella disiens, Moraxella bovoculi (237), Smiihella sp. (SC KO8D17), Leptospira inadai, Lachnospiraceae bacterium (MA2020), Francisella novicida (UI 12), Candidatus Methanoplasma termitum or Eubacterium eligens, or the first complementary domain derived from them.

In particular, the first complementary domain may include an additional base sequence that does not undergo complementary binding to the second complementary domain.

In this case, the additional base sequence may be a sequence of 1-15 bases. For example, the additional base sequence may be a sequence of 1-5, 5-10, or 10-15 bases.

Second complementary domain.

The second complementary domain includes a nucleic acid sequence complementary to the first complementary domain and has a sufficient degree of complementarity to form a double strand with the first complementary domain. The second complementary domain may include a base sequence complementary to the first complementary domain and a base sequence that is not complementary to the first complementary domain, such as a base sequence that does not form a double strand with the first complementary domain and may have a longer base sequence than the first complementary domain. domain.

In this case, the second complementary domain may be or include a sequence of 5-35 bases. For example, the second complementary domain may be a sequence of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 bases.

The second complementary domain may have homology to a naturally occurring second complementary domain or may be derived from a naturally occurring second complementary domain. In addition, the second complementary domain may have a difference in the base sequence of the second complementary domain according to naturally occurring species, may be derived from a second complementary domain found in a naturally occurring species, or may have partial or complete homology to the second complementary domain. domain contained in species existing in nature.

In one embodiment, the second complementary domain may have partial, i.e., at least 50% or greater or complete homology with the second complementary domain

Parcubacteria bacterium (GWC2011_GWC2_44_17), Lachnospiraceae bacterium (MC2017), Butyrivibrio proteoclasiicus, Peregrinibacteria bacterium (GW2011_GWA_33_10), Acidaminococcus sp.

(BV3L6), Porphyromonas macacae, Lachnospiraceae bacterium (ND2006), Porphyromonas crevioricanis, Prevotella disiens, Moraxella bovoculi (237), Smiihella sp. (SCKO8D17), Leptospira inadai, Lachnospiraceae bacterium (MA2020), Francisella novicida (UI 12), Candidatus Methanoplasma termitum or Eubacterium eligens, or a second complementary domain derived from them.

In particular, the second complementary domain may include an additional base sequence that does not undergo complementary binding to the first complementary domain.

In this case, the additional base sequence may be after

- 37 046169 sequence of 1-15 bases. For example, the additional base sequence may be a sequence of 1-5, 5-10, or 10-15 bases.

Linker domain.

Specifically, a linker domain is a nucleic acid sequence connecting a first complementary domain to a second complementary domain to produce a single-stranded gRNA. Here, the linker domain may be connected to the first complementary domain and the second complementary domain by a covalent or non-covalent bond, or may connect the first and second complementary domains by a covalent or non-covalent bond.

The linker domain may be or include a sequence of 1-30 bases. For example, the linker domain may be or include a sequence of 1-5, 5-10, 10-15, 15-20, 20-25 or 25-30 bases.

In particular, part or all of the base sequences of the targeting domain, the first complementary domain, the second complementary domain and the linker domain may be chemically modified. The chemical modification may be methylation, acetylation, phosphorylation, phosphorothioate linkage, locked nucleic acid (LNA), 2'-O-methyl3'-phosphorothioate (MS) or 2'-O-methyl-3'-thioPACE (MSP), with the present invention is not limited to them.

Therefore, a single-stranded gRNA may consist of 5'-[second complementary domain][first complementary domain]-[targeting domain]-3' or 5'-[second complementary domain]-[linker domain]-[first complementary domain]-[ targeting domain]-3' as described above.

In addition, a single-stranded gRNA may selectively include additional base sequence.

In one illustrative embodiment, the single-stranded gRNA may be ^- (Z)h-(Q) ^m_ (N _Mln n _eHb )-3 or

5'-(X) _a -(Z) _h -(X) _b -(Q) _m -(X)c-(Ntarget)“3 .

In another embodiment, the single-stranded gRNA may be

-(Z)h-(L)j-(Q) _m -(N _MmneHb )-3 or

5'-(X) _a -(Z)h-(L)j-(Q) _m -(X) _c -(Ntarget)“3 .

Here, a _target is a base sequence capable of forming a complementary bond with a target sequence in a target gene or nucleic acid, and a region of base sequence that can be changed according to the target sequence in the target gene or nucleic acid.

In one illustrative embodiment, the Hm _target may be a base sequence capable of forming a complementary linkage to a target gene, that is, the target sequence of an unsaturated fatty acid biosynthesis-associated factor, such as a FAD gene, preferably a FAD2 gene, a FAD3 gene, a FAD6 gene, a FAD7 gene or the FAD8 gene.

(Q) _m is a sequence of bases comprising a first complementary domain that can form a complementary bond with a second complementary domain of the second strand. (Q) _m may be a sequence having partial or complete homology with the first complementary domain of a species existing in nature, and the base sequence of the first complementary domain may be changed according to the species of origin. Each Q may be independently selected from the group consisting of A, U, C and G, and m may be a number of bases that is an integer from 5 to 35.

For example, if the first complementary domain has partial or complete homology to the first complementary domain of Parcubacteria bacterium or a first complementary domain derived therefrom, then (Q) _m may be 5'-UUUGUAGAU-3' or a base sequence characterized by at least 50 % or greater homology with 5'-UUUGUAGAU-3'.

(Z)h is a sequence of bases comprising a second complementary domain that can form a complementary bond with the first complementary domain of the first strand. (Z) _h may be a sequence having partial or complete homology with the second complementary domain of a species existing in nature, and the base sequence of the second complementary domain may be modified according to the species of origin. Each Z may be independently selected from the group consisting of A, U, C, and G, and h may be a number of bases that is an integer from 5 to 50.

For example, if the second complementary domain has partial or complete homology to a Parcubacteria bacterium second complementary domain or a Parcubacteria bacterium -derived second complementary domain, then (Z) _h may be

- 38 046169

-AAAUUUCUACU-3 (SEQ ID NO: 46) or a base sequence having at least 50% or greater homology with 5'-AAAUUUCUACU-3' (SEQ ID NO: 46).

In addition, (L)j is a sequence of bases including a linker domain that connects the first complementary domain to the second complementary domain. Each L may be independently selected from the group consisting of A, U, C, and G, and j may be a number of bases that is an integer from 1 to 30.

In addition, each of (X) _a , (X) _b and (X) _c selectively represents an additional sequence of bases, where each X can be independently selected from the group consisting of A, U, C and G, and a, b and c can represent the number of bases, which equals 0 or an integer from 1 to 20.

2. Editing protein.

An editing protein refers to a peptide, polypeptide or protein that can directly bind or interact with a nucleic acid without directly binding to it.

A nucleic acid may be a nucleic acid contained in a target nucleic acid, gene or chromosome.

The nucleic acid may be a guide nucleic acid. The editing protein may be an enzyme. The editing protein may be a fusion protein.

As used herein, the term fusion protein refers to a protein produced by fusing an enzyme with an additional domain, peptide, polypeptide or protein.

An enzyme refers to a protein comprising a domain that is capable of cleaving a nucleic acid, gene, chromosome or protein.

The enzyme may be a nuclease, protease or restriction enzyme.

The additional domain, peptide, polypeptide or protein may be a functional domain, peptide, polypeptide or protein that has the same or different function than the enzyme.

The fusion protein may include an additional domain, peptide, polypeptide, or protein at one or more of the N-terminus of the enzyme or near it, the C-terminus of the enzyme or near it, in the middle region of the enzyme, and combinations thereof.

The fusion protein may include a functional domain, peptide, polypeptide, or protein at one or more of the N-terminus of an enzyme or adjacent to it, the C-terminus of an enzyme or adjacent to it, a mid-region of an enzyme, and combinations thereof.

Here, the functional domain, peptide, polypeptide or protein may be a domain, peptide, polypeptide or protein with methylase activity, demethylase activity, transcription activation activity, transcription repression activity, transcription factor releasing activity, histone modification activity, RNA cleavage activity or activity nucleic acid binding, or a tag or reporter gene for the isolation and purification of a protein (including a peptide), but the present invention is not limited to them.

The functional domain, peptide, polypeptide or protein may be a deaminase.

The tag includes histidine (His) tag, V5 tag, FLAG tag, influenza hemagglutinin (HA) tag, Myc tag, VSV-G tag and thioredoxin (Trx) tag, and the reporter gene includes glutathione S-transferase (GST), horseradish peroxidase (HRP ), chloramphenicol acetyltransferase (CAT) β-galactosidase, β-glucoronidase, luciferase, autofluorescent proteins including green fluorescent protein (GFP), HcRed, DsRed, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP) and blue fluorescent protein (BFP ), but the present invention is not limited to them.

In addition, the functional domain, peptide, polypeptide or protein may be a nuclear localization sequence or signal (NLS) or a nuclear export sequence or signal (NES).

The NLS may be an SV40 large T antigen NLS with the amino acid sequence PKKKRKV (SEQ ID NO: 55); val1elnos1yu

NLS derived from nucleoplasmin (eg, bipartite nucleoplasmin NLS with the sequence KRPAATKKAGQAKKKK) (SEQ ID NO: 56);

c-myc NLS with amino acid sequence PAAKRVKLD RQRRNELKRSP (SEQ ID NO: 58);

(SEQ ID NO: 57) or hRNPAl

M9

NLS

NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ with

ID NO:

sequence 59);

sequence RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO: 60) importin-α-derived IBB domain;

fibroid T protein sequences VSRKRPRP (SEQ ID NO: 61) and PPKKARED (SEQ ID NO: 62);

- 39 046169 sequence PQPKKKPL (SEQ ID NO: 63) human p53; subsequence

SALIKKKKKMAP (SEQ ID NO: 64) c-abl iv _MICE ;

the sequences DRLRR (SEQ ID NO: 65) and PKQKKRK (SEQ ID NO: 66) of the influenza NS1 virus;

sequence RKLKKKIKKL (SEQ ID NO: 67) of hepatitis 5 virus antigen;

sequence REKKKFLKRR (SEQ ID NO: 68) of mouse Mx1 protein;

subsequence

KRKGDEVDGVDEVAKKKSKK (SEQ ID NO: 69) of human poly(ADP-ribose) polymerase or the sequence RKCLQAGMNLEARKTKK (SEQ ID NO: 70) of glucocorticoid steroid hormone receptor (human), but the present invention is not limited to them.

The editing protein may comprise the complete active enzyme.

As used herein, the term fully active enzyme refers to an enzyme having the same function as a wild-type enzyme, and, for example, a wild-type DNA double-strand cleaving enzyme has full enzymatic activity to completely cleave double-strand DNA.

In addition, a fully active enzyme includes an enzyme with improved function over that of the wild-type enzyme, and, for example, a specific type of modification or manipulation of the wild-type double-strand DNA-cleaving enzyme provides complete enzymatic activity that is improved over wild-type enzyme, that is, double-strand DNA cleavage activity.

The editing protein may include an incomplete or partially active enzyme.

As used herein, the term incomplete or partially active enzyme refers to an enzyme that has some of the functions of the wild-type enzyme, and, for example, a specific type of modification or manipulation of the wild-type double-strand DNA cleaving enzyme provides incomplete or partial cleavage enzymatic activity of part of the double strand, that is, a single strand of DNA.

The editing protein may include an inactive enzyme.

As used herein, the term inactive enzyme refers to an enzyme whose wild-type enzyme function is completely inactivated. For example, a specific type of modification or manipulation of the wild-type double-stranded DNA cleaving enzyme has no activity and therefore does not cleave the double-stranded DNA at all.

The editing protein may be a natural enzyme or a fusion protein.

The editing protein may be present in the form of a partially modified natural enzyme or a fusion protein.

The editing protein may be an artificially produced enzyme or a fusion protein that does not exist in nature.

The editing protein may be present in the form of a partially modified artificial enzyme or a fusion protein that does not exist in nature.

In this case, the modification may be the replacement, deletion, addition of amino acids contained in the editing protein, or a combination thereof.

In addition, the modification may be the substitution, deletion, addition of some bases to the base sequence encoding the editing protein, or a combination thereof.

In one illustrative embodiment of the editing protein of the present invention, the CRISPR enzyme will be described below.

CRISPR enzyme.

The term CRISPR enzyme refers to the main protein component of the CRISPR-Cas system that forms a complex with gRNA, giving rise to the CRISPR-Cas system.

A CRISPR enzyme is a nucleic acid or polypeptide (or protein) with a coding sequence for a CRISPR enzyme, and a type II CRISPR enzyme or a type V CRISPR enzyme is widely used as an example.

The type II CRISPR enzyme is Cas9, which can be obtained from various microorganisms, such as

Streptococcus pyogenes, Streptococcus thermophilus, Streptococcus sp., Staphylococcus aureus, Nocardiopsis dassonvillei, Streptomyces pristinaespiralis, Streptomyces viridochromogenes, Streptomyces viridochromogenes, Streptosporangium roseum, Streptosporangium roseum, Alicyclobacillus

- 40 046169 acidocaldarius, Bacillus pseudomycoides, Bacillus selenitireducens. Exiguobacterium sibiricum, Lactobacillus delbrueckii, Lactobacillus salivarius, Microscilla marina, Burkholderiales bacterium, Polaromonas naphthalenivorans, Polaromonas sp., Crocosphaera watsonii, Cyanothece sp., Microcystis aeruginosa, Synechococcus sp., Acetohalobium arabaticum, Ammonifex degensii, Caldic elulosiruptor bescii, Candidatus Desulforudis, Clostridium botulinum , Clostridium difficile, Finegoldia magna, Natranaerobius thermophilus, Pelotomaculum thermopropionicum, Acidithiobacillus caldus, Acidithiobacillus ferrooxidans, Allochromatium vinosum, Marinobacter sp., Nitrosococcus halophilus, Nitrosococcus watsoni, Pseudoalteromonas haloplanktis, Ktedonobacter racemifer, Methanohalobium evestigatum, Anabaena variabilis, Nodularia spumigena, Nostoc sp. , Arthrospira maxima, Arthrospira platensis, Arthrospira sp., Lyngbya sp., Microcoleus chthonoplastes, Oscillatoria sp., Petrotoga mobilis, Thermosipho africanus and Acaryochloris marina.

The term Cas9 means an enzyme that binds to a gRNA and thereby cleaves or modifies a target sequence or location in a target gene or nucleic acid and may consist of an HNH domain capable of cleaving a nucleic acid strand, forming a complementary bond with the gRNA, a RuvC domain capable of cleaving the strand nucleic acid, forming a complementary bond with gRNA, the REC domain that recognizes the target, and the PI domain that recognizes PAM. Hiroshi Nishimasu et al. (2014) Cell 156:935–949 refer to specific structural characteristics of Cas9.

Additionally, the type V CRISPR enzyme may be Cpf1, which may be derived from

Streptococcus, Campylobacter, Nitratifr actor, Staphylococcus, Parvibaculum, Roseburia, Neisseria, Gluconacetobacter, Azospirillum, Sphaerochaeta, Lactobacillus, Eubacterium, Corynebacter, Carnobacterium, Rhodobacter, Listeria, Paludibacter, Clostridium, Lachnospiraceae, Clostridiaridium, Leptotrichia, Francisella, Legionella, Alicyclic obacillus, Methanomethy ophilus, Porphyromonas, Prevotella, Bacteroidetes, Helcococcus, Letospira, De sulfovibrio, Desulfonatronum, Opitutaceae, Tuberibacillus, Bacillus, Brevibacillus, Methylobacterium or Acidaminococcus.

Cpf1 may consist of a RuvC domain similar to and corresponding to the RuvC domain in Cas9, a Nuc domain without the HNH domain in Cas9, a REC domain recognizing the target, a WED domain, and a PI domain recognizing PAMs.

Specific structural characteristics.

Cpf1 can be found in Takashi Yamano et al. (2016) Cell 165:949–962.

The CRISPR enzyme in Cas9 or the Cpf1 protein can be isolated from a naturally occurring microorganism or produced artificially by recombinant or synthetic methods.

Type II CRISPR enzyme.

The crystal structure of the type II CRISPR enzyme was determined according to studies of two or more types of natural microbial type II CRISPR enzyme molecules (Jinek et al., Science, 343(6176): 1247997, 2014) and studies of Streptococcus pyogenes Cas9 (SpCas9) complexed with gRNA (Nishimasu et al., Cell, 156:935–949, 2014; and Anders et al., Nature, 2014, doi: 10.1038/nature13579).

Type II CRISPR enzyme contains two lobes, i.e., recognition lobe (REC) and nuclease lobe (NUC), and each lobe includes several domains.

The REC lobe includes an arginine-rich bridging helix (BH) domain, a REC1 domain, and a REC2 domain.

In this case, the BH domain is a long α-helical and arginine-rich region, and the REC1 and REC2 domains play an important role in recognizing the double strand formed in gRNA, such as single-stranded gRNA, double-stranded gRNA or tracrRNA.

The NUC lobe includes the RuvC domain, the HNH domain, and the PAM interaction (PI) domain. In this case, the RuvC domain encompasses RuvC-like domains, or the HNH domain is used to encompass HNH-like domains.

In this case, the RuvC domain has structural similarity to members of a family of microorganisms that exist in nature, having a type II CRISPR enzyme, and cleaves a single strand, for example, a strand that is not complementary to the target gene or nucleic acid, that is, a strand that does not form a complementary bond with the gRNA . In the prior art, the RuvC domain is sometimes called

- 41 046169 RuvCI domain, RuvCII domain or RuvCIII domain, but is commonly referred to as RuvCI, RuvCII or RuvCIII. For example, in the case of SpCas9, the RuvC domain is assembled from each of three distinct RuvC domains (RuvC I, RuvCII, and RuvCIII) located at amino acid sequences 1–59, 718–769, and 909–1098 in SpCas9, respectively.

The HNH domain has structural similarity to the HNH endonuclease and cleaves a single strand, for example, the complementary strand of the target nucleic acid molecule, that is, the strand forming a complementary bond with the gRNA. The HNH domain is located between RuvC motifs II and III. For example, in the case of SpCas9, the HNH domain is located at amino acid sequence 775-908 in SpCas9.

The PI domain recognizes a specific sequence of bases in a target gene or nucleic acid, that is, a protospacer adjacent motif (PAM), or interacts with PAM. For example, in the case of SpCas9, the PI domain is located in the sequence of amino acids 1099 to 1368 in SpCas9.

In this case, the PAM may vary according to the origin of the type II CRISPR enzyme. For example, if the CRISPR enzyme is SpCas9, then PAM may be 5'-NGG-3', if the CRISPR enzyme is Streptococcus thermophilus Cas9 (StCas9), then PAM may be 5'-NNAGAAW-3'(W=A or T), if the CRISPR enzyme is Neisseria meningitidis Cas9 (NmCas9), then PAM may be 5-NNNNGATT-3', and if the CRISPR enzyme is Campylobacter jejuni Cas9 (CjCas9), then PAM may be 5'NNNVRYAC- 3' (V=G or C or A, R=A or G, Y=C or T), where N may represent A, T, G or C; or A, U, G or C.

Type V CRISPR enzyme.

The type V CRISPR enzyme includes RuvC-like domains corresponding to the RuvC domains of the type II CRISPR enzyme, and may consist of a Nuc domain instead of the HNH domain of the type II CRISPR enzyme, REC and WED domains that recognize the target, and a PI domain that recognizes PAM. Specific structural characteristics of the type V CRISPR enzyme can be found in Takashi Yamano et al. (2016) Cell 165:949–962.

The type V CRISPR enzyme can interact with gRNA, resulting in the formation of a gRNA-CRISPR enzyme complex, that is, the CRISPR complex, and can provide the guide sequence with access to the target sequence, including the PAM sequence together with the gRNA. In this case, the ability of the CRISPR type V enzyme to interact with the target gene or nucleic acid depends on the PAM sequence.

A PAM sequence is a sequence present in a target gene or nucleic acid and can be recognized by the PI domain of a type V CRISPR enzyme. The PAM sequence may vary according to the origin of the type V CRISPR enzyme. That is, there are different PAM sequences that can be specifically recognized depending on the species.

In one example, the PAM sequence that is recognized by Cpf1 may be 5'-TTN-3' (N is A, T, C or G).

CRISPR enzyme activity.

The CRISPR enzyme cleaves the double or single strand of a target gene or nucleic acid and has nuclease activity that causes a break or deletion in the double or single strand. Typically, a wild-type CRISPR type II enzyme or a type V CRISPR enzyme cleaves the double strand of the target gene or nucleic acid.

To manipulate or modify the above-described nuclease activity of a CRISPR enzyme, the CRISPR enzyme may be manipulated or modified, and such manipulated or modified CRISPR enzyme may be modified into an incompletely or partially active or inactive enzyme.

Incompletely or partially active enzyme.

A CRISPR enzyme that has been modified to change enzymatic activity, resulting in incomplete or partial activity, is called nickase.

The term nickase refers to a CRISPR enzyme that has been manipulated or modified to cleave only one strand in a double strand of a target gene or nucleic acid, and wherein nickase has single strand cleavage nuclease activity, e.g., a strand that is not complementary or is complementary to the gRNA of the target gene or nucleic acid.

Therefore, double-strand cleavage requires the nuclease activity of two nickases.

For example, nickase may have nuclease activity due to the RuvC domain. That is, the nickase may have the nuclease activity of the HNH domain, and the HNH domain may be manipulated or modified for this purpose.

In one example, provided that the CRISPR enzyme is a type II CRISPR enzyme, in the case of SpCas9, if residue 840 in the amino acid sequence of SpCas9 is mutated from histidine to alanine, then the nuclease activity of the HNH domain is inactivated to be used as a nickase. Since the nickase thus obtained has the nuclease activity of the RuvC domain, it is able to cleave a strand that does not form a complementary bond with a non-RuvC domain.

- 42 046169 complementary strand of the target gene or nucleic acid, that is, gRNA.

In another exemplary embodiment, in the case of CjCas9, if residue 559 in the amino acid sequence of CjCas9 is mutated from histidine to alanine, the nuclease activity of the HNH domain is inactivated to be used as a nickase. The nickase thus obtained has nuclease activity due to the RuvC domain and is thus capable of cleaving the non-complementary strand of the target gene or nucleic acid, that is, a strand that does not form a complementary bond with the gRNA.

For example, nickase may have nuclease activity due to the HNH domain. That is, the nickase may have the nuclease activity of the RuvC domain, and the RuvC domain may be manipulated or modified for this purpose.

In one example, provided that the CRISPR enzyme is a type II CRISPR enzyme, in one illustrative embodiment, in the case of SpCas9, if residue 10 in the amino acid sequence of SpCas9 is mutated from aspartic acid to alanine, then the nuclease activity of the RuvC domain is inactivated to be used in as a nikase. The nickase thus obtained has the nuclease activity of the HNH domain and is thus capable of cleaving the complementary strand of the target gene or nucleic acid, that is, the strand that forms a complementary bond with the gRNA.

In another exemplary embodiment, in the case of CjCas9, if residue 8 in the amino acid sequence of CjCas9 is mutated from aspartic acid to alanine, then the nuclease activity of the RuvC domain is inactivated to be used as a nickase. The nickase thus obtained has the nuclease activity of the HNH domain and is thus capable of cleaving the complementary strand of the target gene or nucleic acid, that is, the strand that forms a complementary bond with the gRNA.

Inactive enzyme.

A CRISPR enzyme that has been modified to have no enzymatic activity is called an inactive CRISPR enzyme.

The term inactive CRISPR enzyme refers to a CRISPR enzyme that is modified to incompletely cleave the double strand of a target gene or nucleic acid, and wherein the inactive CRISPR enzyme is nuclease inactive due to a mutation in the nuclease activity domain of the wild type CRISPR enzyme. An inactive CRISPR enzyme can be an enzyme in which the nuclease activities of the RuvC domain and the HNH domain are inactivated.

For example, an inactive CRISPR enzyme can be manipulated or modified in the RuvC domain and HNH domain to inactivate nuclease activity.

In one example, provided that the CRISPR enzyme is a type II CRISPR enzyme, in one illustrative embodiment, in the case of SpCas9, if residues 10 and 840 in the amino acid sequence of SpCas9 were mutated from aspartic acid and histidine to alanine, respectively, then nuclease activities due to The RuvC domain and the HNH domain are inactivated, so the double strand cannot completely cleave the double strand of the target gene or nucleic acid.

In another illustrative embodiment, in the case of CjCas9, if residues 8 and 559 in the amino acid sequence of CjCas9 are mutated from aspartic acid and histidine to alanine, then the nuclease activities due to the RuvC domain and the HNH domain are inactivated, so the double strand cannot completely cleave the double strand of targets gene or nucleic acid.

Other activities.

The CRISPR enzyme may have endonuclease activity, exonuclease activity, or helicase activity, that is, it is capable of hybridizing with the helical structure of a double-stranded nucleic acid in addition to the nuclease activity described above.

In addition, the CRISPR enzyme can be modified to completely, incompletely, or partially activate endonuclease activity, exonuclease activity, or helicase activity.

Targeting the CRISPR enzyme.

The CRISPR enzyme can interact with gRNA, due to which the gRNA CRISPR enzyme complex is formed, that is, the CRISPR complex, and provides the guide sequence with access to the target sequence, including the PAM sequence together with the gRNA. In this case, the ability of the CRISPR enzyme to interact with the target gene or nucleic acid depends on the PAM sequence.

A PAM sequence is a sequence present in a target gene or nucleic acid that can be recognized by the PI domain of the CRISPR enzyme. The PAM sequence may vary depending on the origin of the CRISPR enzyme. That is, there are different PAM sequences that can be specifically recognized according to species.

In one example, provided that the CRISPR enzyme is a type II CRISPR enzyme, in the case of SpCas9, the PAM sequence may be 5'-NGG-3', 5'-NAG-3 and/or 5-NGA-3, in the case of StCas9 the PAM sequence may represent

- 43 046169 is 5'-NGGNG-3' and/or 5'-NNAGAAW-3' (W=A or T), in the case of NmCas9 the PAM sequence may be 5'-NNNNGATT-3 and/or 5 -NNNGCTT- 3, in the case of CjCas9, the PAM sequence may be 5'-NNNVRYAC-3' (V=G, C or A; R=A or G; Y=C or T), in the case of Streptococcus mutans Cas9 (SmCas9), the PAM sequence may be 5'-NGG-3' and/or 5'-NAAR-3' (R A or G), and in the case of Staphylococcus aureus Cas9 (SaCas9) the PAM sequence may be 5-NNGRR-3', 5-NNGRRT -3' and/or 5'-NNGRRV-3' (R A or G; V=G, C or A).

As another example, provided that the CRISPR enzyme is a type V CRISPR enzyme, in the case of Cpf 1 the PAM sequence may be 5'-TTN-3'.

Here, N may be A, T, G or C or A, U, G or C.

A CRISPR enzyme capable of recognizing a specific PAM sequence can be manipulated or modified using a PAM sequence that can be specifically recognized according to species. For example, the PI domain of SpCas9 can be replaced with the PI domain of CjCas9 to provide the nuclease activity of SpCas9 and recognition of the CjCas9-specific PAM sequence, thereby obtaining SpCas9 that recognizes the CjCas9-specific PAM sequence. The specifically recognized PAM sequence can be altered by replacement or substitution of the PI domain.

Mutant CRISPR enzyme.

The CRISPR enzyme can be modified to improve or inhibit various characteristics, such as nuclease activity, helicase activity, the ability to interact with gRNA, and the ability to access a target gene or nucleic acid, such as the PAM recognition ability of the CRISPR enzyme.

In addition, a mutant CRISPR enzyme may be a CRISPR enzyme that interacts with gRNA to form a gRNA-CRISPR enzyme complex, i.e., a CRISPR complex, and is modified or manipulated to improve target specificity in reaching or localizing to a target gene or nucleic acid, resulting in cleavage of only the double or single strand of the target gene or nucleic acid without cleavage of the double or single strand of the non-target gene or nucleic acid that partially forms a complementary bond with the gRNA, and of the non-target gene or nucleic acid that does not form a complementary bond with it .

In this case, the effect of double or single strand cleavage of a non-target gene or nucleic acid that partially forms a complementary bond with the gRNA, and a non-target gene or nucleic acid that does not form a complementary bond with it, is called the non-target effect, position or base sequence of the non-target gene or nucleic acid that partially forms a complementary bond with the gRNA, and a non-target gene or nucleic acid that does not form a complementary bond with it are called non-target. In this case, one or more off-target effects may be observed. On the other hand, the effect of double or single strand cleavage of a target gene or nucleic acid is called a targeting effect, and the localization or target sequence of a target gene or nucleic acid is called a target.

The mutant CRISPR enzyme is modified in at least one of the amino acids of the naturally occurring CRISPR enzyme and can be modified, for example, improved or inhibited in one or more of various characteristics, such as nuclease activity, helicase activity, ability to interact with gRNA, ability to reach a target gene or nucleic acid and target specificity, compared to an unmodified CRISPR enzyme. In this case, the modification may be substitution, deletion, addition of an amino acid, or a combination thereof.

In a mutant CRISPR enzyme, the modification may be a modification of one or two or more amino acids located in a region consisting of amino acids with positive charges present in the naturally occurring CRISPR enzyme.

For example, the modification may be a modification of one or two or more amino acids of the positively charged amino acids such as lysine (K), arginine (R) and histidine (H) present in the naturally occurring CRISPR enzyme.

The modification may be a modification of one or two or more amino acids located in a region consisting of non-positively charged amino acids present in the naturally occurring CRISPR enzyme.

For example, the modification may be a modification of one or two or more amino acids from non-positively charged amino acids, i.e., aspartic acid (D), glutamic acid (E), serine (S), threonine (T), asparagine (N), glutamine (Q), cysteine (C), proline (P), glycine (G), alanine (A), valine (V), isoleucine (I), leucine (L), methionine (M), phenylalanine (F), tyrosine (Y) and tryptophan (W) present in the naturally occurring CRISPR enzyme.

As another example, the modification may be a modification of one or two

- 44 046169 or more amino acids from uncharged amino acids, i.e. serine (S), threonine (T), asparagine (N), glutamine (Q), cysteine (C), proline (P), glycine (G), alanine (A) ), valine (V), isoleucine (I), leucine (L), methionine (M), phenylalanine (F), tyrosine (Y), and tryptophan (W), present in the naturally occurring enzyme CRISPR.

In addition, the modification may be a modification of one or two or more amino acids having hydrophobic residues present in a naturally occurring CRISPR enzyme.

For example, the modification may be a modification of one or two or more of the amino acids glycine (G), alanine (A), valine (V), isoleucine (I), leucine (L), methionine (M), phenylalanine (F), tyrosine (Y) and tryptophan (W) present in the naturally occurring CRISPR enzyme.

The modification may be a modification of one or two or more amino acids having polar residues present in a naturally occurring CRISPR enzyme.

For example, the modification may be a modification of one or two or more of the amino acids serine (S), threonine (T), asparagine (N), glutamine (Q), cysteine (C), proline (P), lysine (K), arginine (R), histidine (H), aspartic acid (D) and glutamic acid (E), present in the naturally occurring CRISPR enzyme.

In addition, the modification may be a modification of one or two or more amino acids including lysine (K), arginine (R) and histidine (H) present in the naturally occurring CRISPR enzyme.

For example, the modification may be a substitution of one or two or more amino acids, including lysine (K), arginine (R) and histidine (H), present in the naturally occurring CRISPR enzyme.

The modification may be a modification of one or two or more amino acids, including aspartic acid (D) and glutamic acid (E), present in the naturally occurring CRISPR enzyme.

For example, the modification may be a substitution of one or two or more amino acids, including aspartic acid (D) and glutamic acid (E), present in the naturally occurring CRISPR enzyme.

The modification may be a modification of one or two or more amino acids including serine (S), threonine (T), asparagine (N), glutamine (Q), cysteine (C), proline (P), glycine (G), alanine ( A), valine (V), isoleucine (I), leucine (L), methionine (M), phenylalanine (F), tyrosine (Y) and tryptophan (W), present in the naturally occurring enzyme CRISPR.

For example, the modification may be a substitution of one or two or more amino acids including serine (S), threonine (T), asparagine (N), glutamine (Q), cysteine (C), proline (P), glycine (G), alanine (A), valine (V), isoleucine (I), leucine (L), methionine (M), phenylalanine (F), tyrosine (Y) and tryptophan (W), present in the naturally occurring enzyme CRISPR.

In addition, the modification may be a modification of one, two, three, four, five, six, seven or more amino acids present in a naturally occurring CRISPR enzyme.

Moreover, in a mutant CRISPR enzyme, the modification may be a modification of one or two or more amino acids present in the RuvC domain of the CRISPR enzyme. In this case, the RuvC domain may be a RuvCI, RuvCII, or RuvCIII domain.

The modification may be a modification of one or two or more amino acids present in the HNH domain of the CRISPR enzyme.

The modification may be a modification of one or two or more amino acids present in the REC domain of the CRISPR enzyme.

The modification may be a modification of one or two or more amino acids present in the PI domain of the CRISPR enzyme.

The modification may be a modification of two or more amino acids contained in at least two or more of the REC, RuvC, HNH and PI domains of the CRISPR enzyme.

In one example, the modification may be a modification of two or more amino acids contained in the REC and RuvC domains of the CRISPR enzyme.

In one illustrative embodiment, in a mutant SpCas9, the modification may be a modification of at least two or more of the amino acids A203, H277, G366, F539, I601, M763, D965, and F1038 contained in the REC and RuvC domains of SpCas9.

As another example, the modification may be a modification of two or more amino acids contained in the REC and HNH domains of the CRISPR enzyme.

In one illustrative embodiment, in a mutant SpCas9, the modification may be a modification of at least two or more of amino acids 203, H277, G366, F539, I601, and K890 contained in the REC and HNH domains of SpCas9.

In one example, the modification may be a modification of two or more amino acids contained in the REC and PI domains of the CRISPR enzyme.

- 45 046169

In one illustrative embodiment, in a mutant SpCas9, the modification may be a modification of at least two or more of amino acids 203, H277, G366, F539, I601, T1102, and D1127 contained in the REC and PI domains of SpCas9.

As another example, the modification may be a modification of three or more amino acids contained in the REC, RuvC and HNH domains of the CRISPR enzyme.

In one illustrative embodiment, in a mutant SpCas9, the modification may be a modification of at least three or more of the amino acids A203, H277, G366, F539, I601, M763, K890, D965, and F1038 contained in the REC, RuvC, and HNH domains of SpCas9.

In one example, the modification may be a modification of three or more of the amino acids contained in the REC, RuvC, and PI domains contained in the CRISPR enzyme.

In one illustrative embodiment, in the mutant SpCas9, the modification may be a modification of at least three or more of the amino acids A203, H277, G366, F539, I601, M763, D965, F1038, T1102, and D1127 contained in the REC, RuvC, and PI domains of SpCas9.

As another example, the modification may be a modification of three or more amino acids contained in the REC, HNH and PI domains of the CRISPR enzyme.

In one illustrative embodiment, in a mutant SpCas9, the modification may be a modification of at least three or more of the amino acids A203, H277, G366, F539, I601, K890, T1102, and D1127 contained in the REC, HNH, and PI domains of SpCas9.

In one example, the modification may be a modification of three or more amino acids contained in the RuvC, HNH and PI domains of the CRISPR enzyme.

In one illustrative embodiment, in a mutant SpCas9, the modification may be a modification of at least three or more of the amino acids M763, K890, D965, F1038, T1102, and D1127 contained in the RuvC, HNH, and PI domains of SpCas9.

As another example, the modification may be a modification of four or more amino acids contained in the REC, RuvC, HNH and PI domains of the CRISPR enzyme.

In one illustrative embodiment, in the mutant SpCas9, the modification may be a modification of at least four or more of the amino acids A203, H277, G366, F539, I601, M763, K890, D965, F1038, T1102, and D1127 contained in the REC, RuvC, HNH and PI from SpCas9.

Moreover, in a mutant CRISPR enzyme, the modification may be a modification of one or two or more amino acids involved in the nuclease activity of the CRISPR enzyme.

For example, in a mutant SpCas9, the modification may be a modification of one or two or more groups consisting of amino acids D10, E762, H840, N854, N863 and D986, or one or two or more groups consisting of amino acids corresponding to other Cas9 orthologues.

The modification may be a modification designed to partially inactivate the nuclease activity of the CRISPR enzyme, and such a mutant CRISPR enzyme may be nickase.

In this case, the modification may be a modification designed to inactivate the nuclease activity of the RuvC domain of the CRISPR enzyme, and such mutant CRISPR enzyme may not cleave a non-complementary strand of the target gene or nucleic acid, that is, a strand that does not form a complementary bond with the gRNA.

In one illustrative embodiment, in the case of SpCas9, if residue 10 in the amino acid sequence of SpCas9 has been mutated from aspartic acid to alanine, that is, if a mutation has occurred at D10A, then the nuclease activity of the RuvC domain is inactivated, and thus SpCas9 can be used as a nickase . The nickase thus obtained is not capable of cleaving a non-complementary strand of the target gene or nucleic acid, that is, a strand that does not form a complementary bond with the gRNA.

In another illustrative embodiment, in the case of CjCas9, if residue 8 in the amino acid sequence of CjCas9 has been mutated from aspartic acid to alanine, that is, if a mutation has occurred at D8A, then the nuclease activity of the RuvC domain is inactivated, and thus CjCas9 can be used as a nickase . The nickase thus obtained is not capable of cleaving a non-complementary strand of the target gene or nucleic acid, that is, a strand that does not form a complementary bond with the gRNA.

Moreover, in this case, the modification may be a modification designed to inactivate the nuclease activity of the HNH domain of the CRISPR enzyme, and such mutant CRISPR enzyme is unable to cleave the complementary strand of the target gene or nucleic acid, that is, the strand forming a complementary bond with the gRNA.

In one illustrative embodiment, in the case of SpCas9, if residue 840 in the amino acid sequence of SpCas9 has been mutated from histidine to alanine, that is, if a mutation has occurred at H840A, then the nuclease activity of the HNH domain is inactivated and thus SpCas9 can be used as a nickase. The nikase obtained in this way is not capable of cleaving the complementary strand of the target gene or nucleic acid, that is, the strand that forms the complex

- 46 046169 mental connection with gRNA.

In another exemplary embodiment, in the case of CjCas9, if residue 559 in the amino acid sequence of CjCas9 has been mutated from histidine to alanine, that is, if the mutation at H559A has occurred, then the nuclease activity of the HNH domain is inactivated and thus CjCas9 can be used as a nickase. The nickase thus obtained is not capable of cleaving the complementary strand of the target gene or nucleic acid, that is, the strand that forms a complementary bond with the gRNA.

In addition, the modification may be a modification designed to completely inactivate the nuclease activity of the CRISPR enzyme, and such a mutant CRISPR enzyme may be an inactive CRISPR enzyme.

In this case, the modification may be a modification designed to inactivate the nuclease activities of the RuvC and HNH domains of the CRISPR enzyme, and such mutant CRISPR enzyme cannot cleave the double strand of the target gene or nucleic acid.

In one illustrative embodiment, in the case of SpCas9, if residues 10 and 840 in the amino acid sequence of SpCas9 were mutated from aspartic acid and histidine to alanine, that is, mutated to D10A and H840A, respectively, the nuclease activities of the RuvC domain and the HNH domain are inactivated and the double the target gene or nucleic acid strand cannot be completely cleaved.

In another illustrative embodiment, in the case of CjCas9, if residues 8 and 559 in the amino acid sequence of CjCas9 were mutated from aspartic acid and histidine to alanine, that is, mutated to D8A and H559A, respectively, the nuclease activities of the RuvC and HNH domains are inactivated, and thus the double strand of the target gene or nucleic acid cannot be completely cleaved.

In addition, a mutant CRISPR enzyme may further optionally include a functional domain in addition to the natural characteristics of the CRISPR enzyme, and such a mutant CRISPR enzyme may have an additional characteristic in addition to the natural characteristics.

Here, the functional domain may be a domain with methylase activity, demethylase activity, transcription activation activity, transcription repression activity, transcription factor releasing activity, histone modification activity, RNA cleavage activity or nucleic acid binding activity, or a tag or reporter gene for isolation and protein (including peptide) purification, but the present invention is not limited to them.

The functional domain, peptide, polypeptide or protein may be a deaminase.

For example, an incomplete or partial CRISPR enzyme may additionally include cytidine deaminase as a functional domain. In one illustrative embodiment, a cytidine deaminase, for example, apolipoprotein B editing complex 1 (APOBEC1), can be added to the SpCas9 nickase to thereby produce a fusion protein. The [nickase SpCas9]-[APOBEC1] complex thus formed can be used in base repair or editing of C to T or U or G to A.

The tag includes histidine (His) tag, V5 tag, FLAG tag, influenza hemagglutinin (HA) tag, Myc tag, VSV-G tag and thioredoxin (Trx) tag, and the reporter gene includes glutathione S-transferase (GST), horseradish peroxidase (HRP ), chloramphenicol acetyltransferase (CAT) β-galactosidase, β-glucoronidase, luciferase, autofluorescent proteins including green fluorescent protein (GFP), HcRed, DsRed, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP) and blue fluorescent protein (BFP ), but the present invention is not limited to them.

In addition, the functional domain may be a nuclear localization sequence or signal (NLS) or a nuclear export sequence or signal (NES).

In one example, a CRISPR enzyme may include one or more NLSs. In this case, one or more NLSs may be included at or near the N-terminus of the CRISPR enzyme; The C-terminus of the enzyme or near it or in combination thereof. The NLS may be an NLS sequence derived from the following NLSs, but the present invention is not limited to them:

NLS of the large T antigen of the SV40 virus with the amino acid sequence PKKKRKV (SEQ ID NO: 55);

NLS from nucleoplasmin (eg, bipartite nucleoplasmin NLS with the sequence KRPAATKKAGQAKKKK (SEQ ID NO: 56));

c-myc NLS with the amino acid sequence paakrvkld (SEQ id NO: 57) or RQRRNELKRSP (SEQ ID NO: 58);

hRNPA1 M9 NLS with sequence

NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ ID NO: 59);

- 47 046169 sequence RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO: 60) derived from importin-α IBB domain;

fibroid T protein sequences VSRKRPRP (SEQ ID NO: 61) and PPKKARED (SEQ ID NO: 62);

sequence PQPKKKPL (SEQ ID NO: 63) human p53; subsequence

SALIKKKKKMAP (SEQ ID NO: 64) _with . _aY jv mice;

the sequences DRLRR (SEQ ID NO: 65) and PKQKKRK (SEQ ID NO: 66) of the influenza NS1 virus;

sequence RKLKKKIKKL (SEQ ID NO: 67) of hepatitis delta virus antigen;

sequence REKKKFLKRR (SEQ ID NO: 68) of mouse Mx1 protein;

the sequence KRXGDEVDGVDEVAKKKSKK (SEQ ID NO: 69) of human poly(ADP-ribose) polymerase or NLS with the sequence RKCLQAGMNLEARKTKK (SEQ ID NO: 70) derived from the sequence of the glucocorticoid steroid hormone receptor (human).

In addition, the mutant CRISPR enzyme may include a split-type CRISPR enzyme obtained by dividing the CRISPR enzyme into two or more parts. The term divided refers to the functional or structural division of a protein, or the arbitrary division of a protein into two or more parts.

In this case, the split-type CRISPR enzyme may be a fully, incompletely, or partially active enzyme, or an inactive enzyme.

For example, SpCas9 can be split into two parts between residue 656, tyrosine, and residue 657, threonine, resulting in split SpCas9.

In addition, the split-type CRISPR enzyme may selectively contain an additional domain, peptide, polypeptide or protein for repair.

In this case, recovery refers to the formation of a split-type CRISPR enzyme that must be structurally the same or similar to the wild-type CRISPR enzyme.

The additional repair domain, peptide, polypeptide or protein may be the FRB and FKBP dimerization domains; intein; ERT and VPR domains or domains that form a heterodimer under certain conditions.

For example, SpCas9 can be split into two parts between residue 713, serine, and residue 714, glycine, thereby producing split SpCas9. The FRB domain can be connected to one of the two parts, and the FKBP domain can be connected to the other. In the thus separated SpCas9, the FKB domain and the FKBP domain can be dimerized in an environment in which rapamycin is present, thereby obtaining a reduced CRISPR enzyme.

The CRISPR enzyme or mutant CRISPR enzyme described in the present invention may be a polypeptide, protein or nucleic acid having a coding sequence thereof, and may be codon-optimized to a subject for administration of the CRISPR enzyme or mutant CRISPR enzyme.

The term codon optimization refers to the process of modifying a nucleic acid sequence by maintaining the native amino acid sequence while replacing at least one codon of the native sequence with a codon that is more commonly or most commonly used in host cells to improve expression in the host cells. Different species have a specific preference for a particular codon of a particular amino acid, and codon preference (difference in codon usage between organisms) often correlates with the efficiency of mRNA translation, which is thought to be dependent on the characteristics of the translated codon and the availability of the specific tRNA molecule. The predominance of tRNA selected in cells typically reflects the codons most commonly used in peptide synthesis. Therefore, a gene can be modified by optimally expressing the gene in a given organism based on codon optimization.

3. Target sequence.

The term target sequence is a sequence of bases present in a target gene or nucleic acid and having complementarity with a guide sequence contained in the targeting domain of the guide nucleic acid. The target sequence is a sequence of bases that can vary in accordance with the target gene or nucleic acid, that is, a subject for gene manipulation or correction that can be designed in various forms in accordance with the target gene or nucleic acid.

The target sequence may form a complementary linkage to a guide sequence contained in the targeting domain of the guide nucleic acid, and the length of the target sequence may be the same as that of the guide sequence.

The target sequence may be a sequence of 5-50 bases.

In one embodiment, the target sequence may be a sequence of 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 bases.

The target sequence may be a nucleic acid sequence

- 48 046169 complementary to the targeting sequence contained in the targeting domain of the targeting nucleic acid, which is characterized, for example, by at least 70%, 75%, 80%, 85%, 90% or 95%, or greater complementarity or complete complementarity.

In one example, the target sequence may be or include a sequence of 1-8 bases that is not complementary to a targeting sequence contained in the targeting domain of the guide nucleic acid.

In addition, the target sequence may be a sequence of bases adjacent to a nucleic acid sequence that can be recognized by the editing protein.

In one example, the target sequence may be a contiguous sequence of 5-50 bases adjacent to the 5' end and/or 3' end of a nucleic acid sequence that can be recognized by the editing protein.

In one illustrative embodiment, target sequences for the gRNA-CRISPR enzyme complex will be described below.

When a target gene or nucleic acid is targeted by a gRNA-CRISPR enzyme complex, the target sequence has complementarity to the guide sequence contained in the gRNA guide domain. The target sequence is a sequence of bases that varies in accordance with the target gene or nucleic acid, that is, a subject for gene manipulation or correction that can be designed in various forms in accordance with the target gene or nucleic acid.

In addition, the target sequence may be a base sequence adjacent to a PAM sequence that can be recognized by a CRISPR enzyme, ie Cas9 or Cpf1.

In one example, the target sequence may be a contiguous sequence of 5-50 bases adjacent to the 5' end and/or 3' end of the PAM sequence that is recognized by the CRISPR enzyme.

In one illustrative embodiment, if the CRISPR enzyme is SpCas9, then the target sequence may be a contiguous 16-25 base sequence adjacent to the 5' end and/or 3' end of the 5'-NGG-3' sequence. '-NAG-3' and/or 5'-NGA-3' (N=A, T, G or C; or A, U, G or C).

In another illustrative embodiment, if the CRISPR enzyme is StCas9, then the target sequence may be a contiguous 16-25 base sequence adjacent to the 5' end and/or 3' end of the sequence 5'-NGGNG-3' and/or 5'-NNAGAAW-3' (W=A or T, and N=A, T, G or C; or A, U, G or C).

In the following exemplary embodiment, if the CRISPR enzyme is NmCas9, then the target sequence may be a contiguous 16-25 base sequence adjacent to the 5' end and/or 3' end of the sequence 5'-NNNNGATT-3' and/ or 5'-NNNGCTT-3' (n=A, T, G or C; or A, U, G or C).

In one illustrative embodiment, if the CRISPR enzyme is CjCas9, then the target sequence may be a contiguous 16-25 base sequence adjacent to the 5' end and/or 3' end of the sequence 5'-NNNVRYAC-3' (V =G, C or A; R=A or G, Y=C or T, N=A, T, G or C; or A, U, G or C).

In another illustrative embodiment, if the CRISPR enzyme is SmCas9, then the target sequence may be a contiguous 16-25 base sequence adjacent to the 5' end and/or 3' end of the 5'-NGG-3' and/or sequence. or 5'-NAAR-3' (R=A or G, N=A, T, G or C; or A, U, G or C).

In yet another illustrative embodiment, if the CRISPR enzyme is SaCas9, then the target sequence may be a contiguous 16-25 base sequence adjacent to the 5' end and/or 3' end of the sequence 5-NNGRR-3'. '-NNGRRT-3' and/or 5'-NNGRRV-3' (r=a or G, V=G, C or A, N=A, T, G or C; or A, U, G or C) .

In one illustrative embodiment, if the CRISPR enzyme is Cpf1, the target sequence may be a contiguous 16-25 base sequence adjacent to the 5' end and/or 3' end of the sequence 5'-TTN-3' (N= A, T, G or C; or A, U, G or C).

In one illustrative embodiment of the present invention, the target sequence may be a nucleic acid sequence contained in one or more genes selected from the group consisting of a FAD2 gene, a FAD3 gene, a FAD6 gene, a FAD7 gene, and a FAD8 gene.

The target sequence may be a nucleic acid sequence contained in the FAD2 gene.

- 49 046169

The target sequence may be a nucleic acid sequence contained in the FAD3 gene.

The target sequence may be a nucleic acid sequence contained in the FAD6 gene.

The target sequence may be a nucleic acid sequence contained in the FAD7 gene.

The target sequence may be a nucleic acid sequence contained in the FAD8 gene.

Alternatively, the target sequence may be a partial nucleic acid sequence of one or more genes selected from the group consisting of a FAD2 gene, a FAD3 gene, a FAD6 gene, a FAD7 gene, and a FAD8 gene.

The target sequence may be a partial nucleic acid sequence from the FAD2 gene.

The target sequence may be a partial nucleic acid sequence from the FAD3 gene.

The target sequence may be a partial nucleic acid sequence from the FAD6 gene.

The target sequence may be a partial nucleic acid sequence from the FAD7 gene.

The target sequence may be a partial nucleic acid sequence from the FAD8 gene.

Alternatively, the target sequence may be a coding or non-coding region nucleic acid sequence or a combination thereof of one or more genes selected from the group consisting of a FAD2 gene, a FAD3 gene, a FAD6 gene, a FAD7 gene and a FAD8 gene.

The target sequence may be a coding or non-coding region nucleic acid sequence or a combination thereof from the FAD2 gene.

The target sequence may be a coding or non-coding region nucleic acid sequence or a combination thereof from the FAD3 gene.

The target sequence may be a coding or non-coding region nucleic acid sequence or a combination thereof from the FAD6 gene.

The target sequence may be a coding or non-coding region nucleic acid sequence or a combination thereof from the FAD7 gene.

The target sequence may be a coding or non-coding region nucleic acid sequence or a combination thereof from the FAD8 gene.

Alternatively, the target sequence may be a nucleic acid sequence of a promoter, enhancer, 3'UTR or polyadenylation site (polyA) or a combination thereof of one or more genes selected from the group consisting of FAD2 gene, FAD3 gene, FAD6 gene, FAD7 gene and the FAD8 gene.

The target sequence may be a promoter, enhancer, 3'UTR or polyadenylation site (polyA) nucleic acid sequence or a combination thereof from the FAD2 gene.

The target sequence may be a promoter, enhancer, 3'UTR or polyadenylation site (polyA) nucleic acid sequence or a combination thereof from the FAD3 gene.

The target sequence may be a promoter, enhancer, 3'UTR or polyadenylation site (polyA) nucleic acid sequence or a combination thereof from the FAD6 gene.

The target sequence may be a promoter, enhancer, 3'UTR or polyadenylation site (polyA) nucleic acid sequence or a combination thereof from the FAD7 gene.

The target sequence may be a promoter, enhancer, 3'UTR or polyadenylation site (polyA) nucleic acid sequence or a combination thereof from the FAD8 gene.

Alternatively, the target sequence may be a nucleic acid sequence of an exon, an intron, or a combination thereof of one or more genes selected from the group consisting of a FAD2 gene, a FAD3 gene, a FAD6 gene, a FAD7 gene, and a FAD8 gene.

The target sequence may be the nucleic acid sequence of an exon, intron, or combination thereof of the FAD2 gene.

The target sequence may be the nucleic acid sequence of an exon, intron, or combination thereof of the FAD3 gene.

The target sequence may be a nucleic acid sequence

- 50 046169 exon, intron or combination thereof of the FAD6 gene.

The target sequence may be the nucleic acid sequence of an exon, intron, or combination thereof of the FAD7 gene.

The target sequence may be the nucleic acid sequence of an exon, intron, or combination thereof of the FAD8 gene.

Alternatively, the target sequence may be a nucleic acid sequence comprising or adjacent to a mutated region (eg, a region other than a wild-type gene region) of one or more genes selected from the group consisting of a FAD2 gene, a FAD3 gene, a FAD6, FAD7 gene and FAD8 gene.

The target sequence may be a nucleic acid sequence comprising or adjacent to the mutant region of the FAD2 gene.

The target sequence may be a nucleic acid sequence comprising or adjacent to the mutant region of the FAD3 gene.

The target sequence may be a nucleic acid sequence comprising or adjacent to the mutant region of the FAD6 gene.

The target sequence may be a nucleic acid sequence comprising or adjacent to the mutant region of the FAD7 gene.

The target sequence may be a nucleic acid sequence comprising or adjacent to the mutant region of the FAD8 gene.

Alternatively, the target sequence may be a contiguous nucleic acid sequence of 5 to 50 bases of one or more genes selected from the group consisting of the FAD2 gene, the FAD3 gene, the FAD6 gene, the FAD7 gene, and the FAD8 gene.

The target sequence may be a contiguous 5-50 base nucleic acid sequence of the FAD2 gene.

The target sequence may be a contiguous 5-50 base nucleic acid sequence of the FAD3 gene.

The target sequence may be a contiguous 5-50 base nucleic acid sequence of the FAD6 gene.

The target sequence may be a contiguous 5-50 base nucleic acid sequence of the FAD7 gene.

The target sequence may be a contiguous 5-50 base nucleic acid sequence of the FAD8 gene.

In one illustrative embodiment of the present invention, the above-mentioned FAD2 gene target sequences are summarized in Table. 1.

Product of a manipulable factor associated with the biosynthesis of unsaturated fatty acids.

4. Complex guide nucleic acid - editing protein and its application.

The targeting nucleic acid-editing protein complex can modify the target.

For example, a targeting nucleic acid-editing protein complex can be used to maximally regulate (e.g., inhibit, suppress, reduce, enhance, or stimulate) the expression of a protein of interest, remove the protein, regulate (e.g., inhibit, suppress, reduce, enhance, or stimulate) the expression of a protein of interest. protein activity or new protein expression.

In this case, the guiding nucleic acid-editing protein complex can act at the level of DNA, RNA, gene or chromosome.

For example, a guide nucleic acid-editing protein complex may regulate (e.g., inhibit, suppress, decrease, increase, or stimulate) the expression of a protein encoded by the target DNA, remove a protein, regulate (e.g., inhibit, suppress, decrease, increase, or stimulate) the activity of a protein or express a modified protein through manipulation or modification of the target DNA.

In another example, the guide nucleic acid-editing protein complex may regulate (e.g., inhibit, suppress, decrease, increase, or stimulate) the expression of a protein encoded by the target DNA, remove the protein, regulate (e.g., inhibit, suppress, decrease, increase, or stimulate) the activity protein or express a modified protein by manipulating or modifying the target RNA.

In one example, the guide nucleic acid-editing protein complex may regulate (e.g., inhibit, suppress, decrease, increase, or stimulate) the expression of a protein encoded by the target DNA, remove the protein, regulate (e.g., inhibit, suppress, decrease, increase, or stimulate) the activity protein or express a modified protein through manipulation or modification of a target gene.

As another example, a guide nucleic acid-editing protein complex

- 51 046169 can regulate (eg, inhibit, suppress, reduce, increase, or stimulate) the expression of a protein encoded by the target DNA, remove a protein, regulate (eg, inhibit, suppress, reduce, increase, or stimulate) the activity of a protein, or express a modified protein by manipulation or modifications of the target chromosome.

The guiding nucleic acid-editing protein complex can act at the stages of gene transcription and translation.

In one example, the guide nucleic acid-editing protein complex can stimulate or repress transcription of a target gene, thereby regulating (eg, inhibiting, suppressing, reducing, increasing, or stimulating) the expression of the protein encoded by the target gene.

In another example, the guide nucleic acid-editing protein complex may stimulate or inhibit translation of a target gene, thereby regulating (eg, inhibiting, suppressing, reducing, increasing, or stimulating) the expression of the protein encoded by the target gene.

The guide nucleic acid-editing protein complex can act at the protein level.

In one example, the targeting nucleic acid-editing protein complex can manipulate or modify a target protein, thereby removing the target protein or regulating (eg, inhibiting, suppressing, reducing, increasing, or stimulating) the activity of the protein.

In one illustrative embodiment, the present invention provides a guide nucleic acid-editing protein complex used to manipulate an unsaturated fatty acid biosynthesis-associated factor, for example, the FAD gene, preferably the FAD2 gene, the FAD3 gene, the FAD6 gene, the FAD7 gene and/or FAD8 genome. Preferably, a gRNA-CRISPR enzyme complex is provided.

In particular, the present invention may provide a gRNA including a targeting domain capable of forming a complementary linkage to a target sequence from a gene, for example, an isolated or non-naturally occurring gRNA and DNA encoding it. The gRNA and the DNA sequence encoding it can be designed to bind complementarily to the target sequence shown in Table 1. 1.

In addition, the gRNA target region is designed to provide a third gene that has a nucleic acid modification, such as double or single strand breaks; or a specific function at a target site in the FAD2 gene, FAD3 gene, FAD6 gene, FAD7 gene and/or FAD8 gene.

Additionally, when two or more gRNAs are used to induce two or more cleavage events in a target gene, such as double or single strand breaks, two or more cleavage events may occur due to the same or different Cas9 proteins.

The gRNA may target, for example, two or more of the FAD2 gene, the FAD3 gene, the FAD6 gene, the FAD7 gene, and/or the FAD8 gene, or two or more regions in each of the FAD2 gene, the FAD3 gene, the FAD6 gene, the FAD7 gene, and/or or the FAD8 gene, and can independently induce double-strand and/or single-strand cleavage of the FAD2 gene, FAD3 gene, FAD6 gene, FAD7 gene and/or FAD8 gene, or can induce the insertion of a single foreign nucleotide into the cleavage site of the FAD2 gene, FAD3 gene, gene FAD6, FAD7 gene and/or FAD8 gene.

Additionally, in another exemplary embodiment of the present invention, the nucleic acid that makes up the guide nucleic acid-editing protein complex may include:

(a) a sequence encoding a targeting nucleic acid including a targeting domain that is complementary to the target sequence of the FAD2 gene as described herein; and (b) an editing protein coding sequence.

Here, two or more of (a) may be present according to the target site, and (b) the same or two or more editing proteins may be used.

In one embodiment, the nucleic acid can be designed to target an enzymatically inactive editing protein or a fusion protein thereof (e.g., a transcriptional repressor domain fusion) to position it sufficiently close to the target knockdown site to reduce, reduce, or inhibit FAD2 gene expression.

Moreover, it should be apparent that the above-described structure, function and all uses of the guide nucleic acid-editing protein complex will be used in the manipulation of the FAD2 gene, FAD3 gene, FAD6 gene, FAD7 gene and/or FAD8 gene.

Application of a guide nucleic acid-editing protein complex.

In one embodiment, for the use of a guide nucleic acid editing protein complex in accordance with the present invention, the manipulation or modification of target DNA, RNA, genes or chromosomes using the gRNA complex will be described below.

- 52 046169 CRISPR enzyme.

Gene manipulation.

The target gene or nucleic acid can be manipulated or corrected using the gRNA-CRISPR enzyme complex described above, that is, the CRISPR complex. Here, manipulation or correction of the target gene or nucleic acid includes the steps of i) cleavage or damage to the target gene or nucleic acid and ii) repair of the damaged target gene or nucleic acid.

i) Cleavage or damage to the target gene or nucleic acid.

i) Cleavage or damage to a target gene or nucleic acid may be cleavage or damage to a target gene or nucleic acid using a CRISPR complex, and in particular, cleavage or damage to a target sequence in a target gene or nucleic acid.

In one example, cleavage or damage to a target gene or nucleic acid using a CRISPR complex may be a complete double strand cleavage or damage of the target sequence.

In one exemplary embodiment, when using wild-type SpCas9, the double strand of the target sequence forming a complementary bond to the gRNA can be completely cleaved.

In another exemplary embodiment, when using SpCas9 (D10A) nickase and SpCas9 (H840A) nickase, the complementary single strand of the target sequence forming a complementary linkage to the gRNA can be cleaved by the SpCas9 (D10A) nickase, rather than the non-complementary single strand of the target sequence forming the complementary bound to gRNA, can be cleaved by SpCas9 (H840A) nickase, and the cleavages can occur sequentially or simultaneously.

In the following exemplary embodiment, when using SpCas9 nickase (D10A), SpCas9 nickase (H840A) and two gRNAs having different target sequences, the complementary single strand of the target sequence forming a complementary linkage to the first gRNA can be cleaved by SpCas9 nickase (D10A) without the complementary single strand of the target sequence forming a complementary linkage to the second gRNS can be cleaved by SpCas9 (H840A) nickase, and the cleavages can occur sequentially or simultaneously.

As another example, cleavage or damage to a target gene or nucleic acid using a CRISPR complex may be cleavage or damage to only a single strand of the target sequence. Here, the single strand may be a complementary single strand of the target sequence forming a complementary linkage to the gRNA or a non-complementary single strand of the target sequence forming a complementary linkage to the gRNA.

In one illustrative embodiment, when using SpCas9 (D10A) nickase, the complementary single strand of the target sequence forming a complementary linkage to the gRNA can be cleaved by SpCas9 nickase (D1OA), but the non-complementary single strand of the target sequence forming a complementary linkage to the gRNA cannot be split.

In another exemplary embodiment, when using SpCas9 (H840A) nickase, the complementary single strand of the target sequence forming a complementary linkage to the gRNA can be cleaved by SpCas9 (H840A) nickase, but the non-complementary single strand of the target sequence forming a complementary linkage to the gRNA cannot be split.

As another example, cleavage or damage to a target gene or nucleic acid using a CRISPR complex may involve partial removal of a fragment of the nucleic acid.

In one illustrative embodiment, when using two gRNAs having different target sequences and wild-type SpCas9, the double strand of the target sequence forming a complementary linkage to the first gRNA can be cleaved, and the double strand of the target sequence forming a complementary linkage to the second gRNA can be is cleaved, which leads to the removal of nucleic acid fragments due to the first and second gRNA and SpCas9.

In another illustrative embodiment, when using two gRNAs having different target sequences, wild-type SpCas9, SpCas9 nickase (D10A) and SpCas9 nickase (H840A), the double strand of the target sequence forming a complementary linkage to the first gRNA can be cleaved by wild-type SpCas9, complementary the single strand of the target sequence forming a complementary linkage to the second gRNA can be cleaved by SpCas9 nickase (D10A), and the non-complementary single strand can be cleaved by SpCas9 nickase (H840A), resulting in the removal of nucleic acid fragments by the first and second gRNA, Wild-type SpCas9, SpCas9 nickase (D10A), and SpCas9 nickase (H840A).

- 53 046169

In the following exemplary embodiment, when using two gRNAs having different target sequences, SpCas9 (D10A) nickase and SpCas9 (H840A) nickase, the complementary single strand of the target sequence forming a complementary linkage to the first gRNA can be cleaved by the SpCas9 (D10A) nickase that is not a complementary single strand can be cleaved by SpCas9 nickase (H840A), a complementary double strand of a target sequence forming a complementary linkage to a second gRNA can be cleaved by SpCas9 nickase (D10A), and a non-complementary single strand can be cleaved by SpCas9 nickase (H840A), which leads to the removal of nucleic acid fragments by the first and second gRNAs, SpCas9 (D10A) nickase and SpCas9 (H840A) nickase.

In yet another exemplary embodiment, when using three gRNAs having different target sequences, wild-type SpCas9, SpCas9 nickase (D10A), and SpCas9 nickase (H840A), the double strand of the target sequence forming a complementary linkage to the first gRNA can be cleaved by wild-type SpCas9, the complementary single strand of a target sequence forming a complementary linkage to a second gRNA can be cleaved by SpCas9 nickase (D10A), and the non-complementary single strand of a target sequence forming a complementary linkage to a third gRNA can be cleaved by SpCas9 nickase (H840A), resulting in removal of nucleic acid fragments using the first gRNA, second gRNA, third gRNA, wild-type SpCas9, SpCas9 nickase (D10A) and SpCas9 nickase (H840A).

In yet another exemplary embodiment, when using four gRNAs having different target sequences, SpCas9 nickase (D10A) and SpCas9 nickase (H840A), the complementary single strand of the target sequence forming a complementary linkage to the first gRNA can be cleaved by SpCas9 nickase (D10A) without the complementary single strand of a target sequence forming a complementary linkage to a second gRNA can be cleaved by SpCas9 (H840A) nickase, the complementary single strand of a target sequence forming a complementary linkage to a third gRNA can be cleaved by SpCas9 nickase (D10A), and a non-complementary single strand The strand of the target sequence forming a complementary bond with the fourth gRNA can be cleaved by SpCas9 nickase (H840A), resulting in the removal of nucleic acid fragments by the first gRNA, second gRNA, third gRNA, fourth gRNA, SpCas9 nickase (D10A) and SpCas9 nickase ( H840A).

ii) Repair or restoration of damaged target gene or nucleic acid.

A target gene or nucleic acid cleaved or damaged by the CRISPR complex can be repaired or restored using non-homologous end joining (NHEJ) and homology-directed repair (HDR).

Non-homologous end joining (NHEJ).

NHEJ is a method of repairing double-strand breaks in DNA by joining together both ends of a split double or single strand and, typically, if the two compatible ends formed by the double-strand break (such as a cleavage) are in frequent contact with each other to completely connect the two ends, the broken double thread is restored. NHEJ is a repair pathway that can be used throughout the cell cycle, but is typically performed when there is no homologous genome to use as a template in cells, such as in G1 phase.

During the process of repair of a damaged gene or nucleic acid using NHEJ, some insertions and/or deletions (insertion-deletion mutations) in the nucleic acid sequence are carried out in the region repaired using NHEJ, and such insertions and/or deletions cause a shift of the leading frame, which gives Frameshifted mRNA transcriptome. As a result, natural functions are lost due to nonsense-mediated degradation or the inability to synthesize normal proteins. In addition, although the leading frame is conserved, mutations in which a significant amount of insertion or deletion in the sequence can be introduced to impair the functionality of the proteins. The mutation is locus dependent because a mutation in a significant functional domain is likely to be less tolerated than mutations in a non-significant region of the protein.

Although NHEJ-derived insertion-deletion mutations cannot be expected to occur in the natural state, the specific sequence with the insertion-deletion mutation is favored at a given disrupted site and may originate from a small region of microhomology. Typically, the length of the deletion varies from 1 bp. up to 50 bp, insertions tend to be shortened and often include a short sequence of repeats immediately surrounding the disrupted region.

In addition, NHEJ is a mutation-inducing process and, if there is no need to create a specific resulting sequence, it can be used to remove a small sequence motif.

Using such NHEJ, specific knockout of the gene targeted by the CRISPR complex can be performed. Double strand or two single strands of the target gene or nucleic acid

- 54 046169 acids can be cleaved using a CRISPR enzyme such as Cas9 or Cpfl, and the double strand or two single strands of the target gene or nucleic acid with the disorder can have insertion-deletion mutations due to NHEJ, thereby inducing a specific knockout of the target gene or nucleic acid. In this case, the site of the target gene or nucleic acid cleaved by the CRISPR enzyme may be a non-coding or coding region, and further, the site of the target gene or nucleic acid recovered by NHEJ may be a non-coding or coding region .

Homology-directed repair (HDR).

HDR is an error-free correction method that uses a homologous sequence as a template to repair or restore a damaged gene or nucleic acid and, as a rule, to repair or restore damaged DNA, that is, to restore the hereditary information of cells, while the damaged DNA repaired using information from a complementary base sequence that is not modified, or information from a sister chromatid. The most common type of HDR is homologous recombination (HR). HDR is a mode of repair or restoration that typically occurs in the S or G2/M phase of actively dividing cells.

To repair or restore damaged DNA using HDR, instead of using the complementary base sequence or sister chromatin of cells, a DNA template artificially synthesized using information from the complementary base sequence or homologous base sequence, that is, a nucleic acid template including a complementary base sequence or homologous base sequence, can be provided in cells with the repair of thereby damaged DNA. In this case, when a nucleic acid sequence or nucleic acid fragment is further added to a nucleic acid template for repairing damaged DNA, the nucleic acid sequence or nucleic acid fragment further added to the damaged DNA may be knocked out. Additionally, the added nucleic acid sequence or nucleic acid fragment may be a nucleic acid sequence or nucleic acid fragment intended to correct the target gene or nucleic acid modified by mutation to a normal gene or nucleic acid or gene or nucleic acid to be expressed in cells, however, the present invention is not limited to them.

In one example, a double or single strand of a target gene or nucleic acid may be cleaved using a CRISPR complex, wherein a nucleic acid template may be provided in cells comprising a base sequence complementary to a base sequence adjacent to the cleavage site, and thereby cleaved the base sequence of the target gene or nucleic acid can be repaired or restored by HDR.

Here, the nucleic acid template including the complementary base sequence may have disrupted DNA, that is, a split double or single strand of the complementary base sequence, and further include a nucleic acid sequence or nucleic acid fragment to be inserted into the disrupted DNA. An additional nucleic acid sequence or nucleic acid fragment can be inserted into a cleavage site of disrupted DNA, ie, a target gene or nucleic acid, using a nucleic acid template comprising the nucleic acid sequence or nucleic acid fragment to be inserted into a complementary base sequence. Here, the nucleic acid sequence or nucleic acid fragment to be inserted, and the additional nucleic acid sequence or nucleic acid fragment may be a nucleic acid sequence or nucleic acid fragment for correcting a target gene or nucleic acid modified by mutation to a normal gene or a nucleic acid, or a gene or nucleic acid, to be expressed in cells. A complementary base sequence may be a base sequence having complementary bonds with the disrupted DNA, that is, with the right and left base sequences of the split double or single strand of the target gene or nucleic acid. Alternatively, the complementary base sequence may be a base sequence having complementary bonds with the disrupted DNA, that is, the 3' and 5' ends of the split double or single strand of the target gene or nucleic acid. The complementary base sequence may be a sequence of 15 to 3000 bases, and the length or size of the complementary base sequence may be suitably designed according to the size of the nucleic acid template or the target gene. In this case, the nucleic acid template may be a double or single stranded nucleic acid, or it may be linear or circular, but the present invention is not limited to

- 55 046169 them.

As another example, a double- or single-stranded target gene or nucleic acid is cleaved using a CRISPR complex, wherein a nucleic acid template comprising a base sequence homologous to a base sequence adjacent to the cleavage site can be provided in cells, and the cleaved sequence bases of the target gene or nucleic acid can be repaired or restored by HDR.

Here, the nucleic acid template including the homologous base sequence may be a disrupted DNA, that is, a split double- or single-stranded homologous base sequence, and further includes a nucleic acid sequence or a nucleic acid fragment to be inserted into the disrupted DNA. Additional nucleic acid sequence or nucleic acid fragment can be inserted into the disrupted DNA, that is, the cleavage site of the target gene or nucleic acid, using a nucleic acid template including a homologous base sequence and the nucleic acid sequence or nucleic acid fragment to be inserted. Here, the nucleic acid sequence or nucleic acid fragment to be inserted, and the additional nucleic acid sequence or nucleic acid fragment may be a nucleic acid sequence or nucleic acid fragment for correcting a target gene or nucleic acid modified by mutation to a normal gene or a nucleic acid, or a gene or nucleic acid, to be expressed in cells. The homologous base sequence may be a disrupted DNA sequence, that is, a base sequence having homology with the split double-stranded base sequence or the right and left single-stranded base sequences of the target gene or nucleic acid. Alternatively, the complementary base sequence may be a base sequence having homology to the disrupted DNA, that is, to the 3' and 5' ends of the split double or single strand of the target gene or nucleic acid. The homologous base sequence may be a sequence of 15 to 3000 bases, and the length or size of the homologous base sequence may be suitably designed according to the size of the template in the form of a nucleic acid or a target gene or nucleic acid. In this case, the nucleic acid template may be a double- or single-stranded nucleic acid, which may be linear or circular, but the present invention is not limited to them.

In addition to NHEJ and HDR, there are methods to repair or restore damaged DNA.

Single-strand hybridization (SSA).

SSA is a method for repairing double strand breaks between two repeat sequences present in a target nucleic acid and typically uses a repeat sequence of more than 30 bases. The repeat sequence is cleaved (with sticky ends) to produce a single strand to a double strand of the target nucleic acid at each disrupted end, and after cleavage, the single strand overhang containing the repeat sequence is coated with RPA protein, thereby preventing misfolding. hybridization of repeat sequences with each other. RAD52 binds to each repeat sequence at the overhang and establishes a sequence capable of hybridizing to the complementary repeat sequence. After hybridization, the single-stranded overhanging portion of the overhang is cleaved and new DNA synthesis involves filling a specific gap to restore the double strand of DNA. This repair removes the DNA sequence between the two repeats, and the length of the deletion may depend on various factors, including the location of the two repeats used herein and the path or extent of cleavage.

SSA, similar to HDR, uses a complementary sequence, that is, a complementary repeat sequence, but does not require a nucleic acid template to modify or correct the target nucleic acid sequence for comparison.

Single-strand break repair (SSBA).

Single-strand breaks in the genome are repaired by a separate mechanism, SSBR, from the repair mechanisms described above. In the case of single-strand DNA breaks, PARP1 and/or PARP2 recognizes the breaks and engages the repair mechanism. PARP1 binding and DNA break activity are transient and SSBR is stimulated by maintaining the stability of the SSBR protein complex at damaged sites. The most important protein in the SSBR complex is XRCC1, which interacts with the protein to activate 3' and 5' DNA processing to stabilize DNA. Terminal processing is typically involved in the repair of a damaged 3' end to a hydroxylated state and/or a damaged 5' end to a phosphate moiety, and after the process

- 56 046169 end siding occurs when DNA gaps are filled. There are two ways to fill DNA gaps, that is, short sequence repair and long sequence repair, and short sequence repair involves the insertion of a single base. Once the DNA gap is filled, DNA ligase ensures that the ends are joined.

Mismatch repair (MMR).

MMR works with mismatched DNA bases. Each of the MSH2/6 or MSH2/3 complexes has ATPase activity and thus plays an important role in mismatch recognition and initiation of repair, with MSH2/6 primarily recognizing base-to-base mismatches and identifying single or double base mismatches, a MSH2/3 mainly recognizes larger mismatches.

Base excision repair (BER).

BER is a repair pathway that is active throughout the cell cycle and is used to remove small, non-helical base damage from the genome. From damaged DNA, damaged bases are removed by cleavage of the N-glycosidic bond connecting the base to the phosphate-deoxyribose backbone, and then the phosphodiester backbone is cleaved, thereby creating a break in the single-stranded DNA. The broken ends of the single strand thus formed are removed, the gap created by the removed single strand is filled with a new complementary base, and then the end of the newly filled complementary base is ligated to the scaffold by DNA ligase, resulting in repair of the damaged DNA.

Nucleotide excision repair (NER)

NER is an excision mechanism that plays an important role in removing large helix-disrupting lesions from DNA, where damage recognition removes a short single-stranded segment of DNA containing the damaged region, resulting in a single-stranded skip of 22-30 bases. The resulting gap is filled with a new complementary base, and the end of the newly filled complementary base is ligated to the framework using DNA ligase, due to which the damaged DNA is repaired.

Effects of gene manipulation.

Manipulation or correction of a target gene or nucleic acid can significantly lead to knockout, knockdown, and knockout effects.

Knockout.

The term knockout refers to the inactivation of a target gene or nucleic acid, and the term inactivation of a target gene or nucleic acid refers to a condition in which transcription and/or translation of a target gene or nucleic acid does not occur. Transcription and translation of a disease-causing gene or a gene with impaired function can be inhibited by knockout, resulting in the prevention of protein expression.

For example, when editing or correcting a target gene or nucleic acid using a gRNA-CRISPR enzyme complex, that is, a CRISPR complex, the target gene or nucleic acid can be cleaved using the CRISPR complex. Damaged target gene or nucleic acid can be repaired by NHEJ using the CRISPR complex. The damaged target gene or nucleic acid may have insertion-deletion mutations generated by NHEJ, and thereby a specific knockout of the target gene or nucleic acid can be induced.

Knockdown.

The term knockdown refers to a reduction in the transcription and/or translation of a target gene or nucleic acid or the expression of a target protein. The occurrence of a disease can be prevented or the disease can be cured by regulating the overexpression of a gene or protein through knockdown.

For example, when editing or correcting a target gene or nucleic acid using a gRNA-inactive enzyme CRISPR-transcription inhibitory activity domain complex, that is, an inactive CRISPR complex including a transcription inhibitory activity domain, the inactive CRISPR complex may specifically bind to the target gene or nucleic acid, transcription of a target gene or nucleic acid can be inhibited by a transcription inhibitory activity domain included in the inactive CRISPR complex, thereby inducing a knockdown in which expression of the corresponding gene or nucleic acid is inhibited.

Knockin.

The term knockin refers to the insertion of a specific nucleic acid or gene into a target gene or nucleic acid, and as used herein, the term specific nucleic acid refers to the gene or nucleic acid of interest to be inserted or expressed. A disease-triggering mutant gene can be used to treat the disease by correcting it to a normal gene or inserting a normal gene to induce expression of the normal gene by

- 57 046169 by knockin.

In addition, knockin may additionally require a donor.

For example, when editing or correcting a target gene or nucleic acid using a gRNA-CRISPR enzyme complex, that is, a CRISPR complex, the target gene or nucleic acid can be cleaved using the CRISPR complex. A target gene or nucleic acid damaged by the CRISPR complex can be repaired through HDR. In this case, a specific nucleic acid can be inserted into the damaged gene or nucleic acid using a donor.

The term donor refers to a nucleic acid sequence that promotes HDR-based repair of a damaged gene or nucleic acid, and in this case the donor may include a specific nucleic acid.

The donor can be a double or single stranded nucleic acid. The donor may be present in linear or ring form.

The donor may include a nucleic acid sequence having homology to the target gene or nucleic acid.

For example, the donor may include a nucleic acid sequence having homology to each of the base sequences at locations in which the specific nucleic acid is to be inserted, for example, in the 3'-5' direction (left) and 5'-3' direction (right ) damaged nucleic acid. In this case, the specific nucleic acid to be inserted may be located between a nucleic acid sequence having homology with a base sequence in the 5'-3 direction of the damaged nucleic acid and a nucleic acid sequence having homology with a base sequence in the 3'-5' direction -direction of damaged nucleic acid. Here, the homologous nucleic acid sequence may have at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% or greater homology or complete homology.

The donor may optionally include an additional nucleic acid sequence. In this case, the additional nucleic acid sequence may serve to enhance donor stability, knockin efficiency, or HDR efficiency.

For example, the additional nucleic acid sequence may be an A-rich nucleic acid sequence, that is, an A-T rich domain. In addition, the additional nucleic acid sequence may be a backbone/template attachment region (SMAR).

In one exemplary embodiment related to the effect of gene manipulation in accordance with the present invention, the manipulated target gene obtained using the gRNA-CRISPR enzyme complex, that is, the manipulated unsaturated fatty acid biosynthesis associated factor, may have the following structure.

In one illustrative embodiment, if the unsaturated fatty acid biosynthesis-associated factor is a gene, then the structure of the unsaturated fatty acid biosynthesis-associated factor manipulated by the gRNA-CRISPR enzyme complex may include modification of one or more nucleic acids along with deletion or insertion of one or more nucleotides; substitution with one or more nucleotides other than those of a wild-type gene and insertion of one or more foreign nucleotides within a contiguous region of 1 bp in length. up to 50 bp, from 1 bp up to 40 p.o. or from 1 p.o. up to 30 bp, preferably from 3 bp up to 25 p.o. in a base sequence that is located in the PAM sequence within the nucleic acid sequence of which the unsaturated fatty acid biosynthesis-associated factor is composed or adjacent to its 5' end and/or 3' end.

In addition, a chemical modification of one or more nucleotides may be included in the nucleic acid sequence of which the unsaturated fatty acid biosynthesis-associated factor is composed.

As used herein, the term foreign nucleotide is a concept that includes all exogenous, eg, heterologous or artificially synthesized nucleotides other than the nucleotides naturally included in the unsaturated fatty acid biosynthesis-associated factor. A foreign nucleotide also includes a nucleotide of several hundred, thousand, or tens of thousands of bp in size designed to express a protein having a specific function, as well as a small oligonucleotide of 50 bp in size. or less. Such a foreign nucleotide can be a donor.

Chemical modification may include methylation, acetylation, phosphorylation, ubiquitinylation, ADP-ribosylation, myristylation and glycosylation, such as replacing several functional groups contained in a nucleotide with any of a hydrogen atom, a fluorine atom, an -O-alkyl group, an -O-acyl group and amino groups, but the present invention is not limited to them. In addition, to enhance the transfer ability of the nucleic acid molecule, functional groups can also be replaced by any of -Br, -Cl, -R, -R'OR, -SH, -SR, -N ₃ and -CN (I = alkyl. aryl, alkylene). In addition, the phosphate backbone of at least one nucleotide may

- 58 046169 be substituted by any of the alkylphosphonate form, phosphoramidate form and boranophosphate form. Moreover, the chemical modification may be the replacement of at least one type of nucleotide contained in a nucleic acid molecule with any of a locked nucleic acid (LNA), an unlocked nucleic acid (UNA), a morpholino, and a peptide nucleic acid (PNA), and wherein the chemical modification may be the binding of a nucleic acid molecule to one or more selected from the group consisting of a lipid, a cell penetrating peptide, and a cell targeting ligand.

To obtain the required system for controlling the biosynthesis of unsaturated fatty acids, artificial modification using the gRNA-enzyme complex CRISPR can be applied to the nucleic acid that makes up the factor associated with the biosynthesis of unsaturated fatty acids.

The region containing the nucleic acid modification of the unsaturated fatty acid biosynthesis-associated factor may be a target region or a target sequence.

Such a target sequence may be the target of a gRNA-CRISPR enzyme complex, and the target sequence may or may not include a PAM sequence recognized by the CRISPR enzyme. Such a target sequence can provide a critical standard during gRNA design for those skilled in the art.

Such modification of a nucleic acid involves cleavage of the nucleic acid.

The term target site cleavage refers to the cleavage of the covalent backbone of polynucleotides. Cleavage involves enzymatic or chemical hydrolysis of the phosphodiester bond, but the present invention is not limited to these, but also includes various other methods. Splitting can be performed on either a single strand or a double strand, and splitting on a double strand can result from the splitting of individual single strands. Double strand splitting may produce blunt ends or stepped ends.

By using an inactivated CRISPR enzyme, it is possible to induce a factor that has a specific function to reach a certain site in the target site or a factor associated with the biosynthesis of unsaturated fatty acids without a cleavage process. Chemical modification of one or more nucleotides in the nucleic acid sequence of the unsaturated fatty acid biosynthesis-associated factor may be included in accordance with such specific function.

In one example, various insertion-deletion mutations may occur due to on-target and off-target activities through nucleic acid cleavage carried out by the gRNA-CRISPR enzyme complex.

The term insertion-deletion mutation is a general term for an insertion or deletion mutation occurring between certain bases in a sequence of DNA bases. An insertion-deletion mutation can be introduced into the target sequence during repair by the HDR or NHEJ mechanism if the gRNA-CRISPR enzyme complex cleaves the nucleic acid (DNA or RNA) associated with the biosynthesis of unsaturated fatty acids, as described above.

The artificially manipulated unsaturated fatty acid biosynthesis-associated factor of the present invention relates to modification of the nucleic acid sequence of the original gene by cleavage, insertion-deletion mutations or insertion using a donor such as nucleic acid and contributes to the required control system biosynthesis of unsaturated fatty acids, for example, demonstrating the effect of promoting or inhibiting a specific unsaturated fatty acid.

For example, a specific protein may be expressed and its activity may be stimulated by an artificially manipulated unsaturated fatty acid biosynthesis-associated factor.

A specific protein can be inactivated by a factor associated with the biosynthesis of unsaturated fatty acids.

In one example, a specific target region of each unsaturated fatty acid biosynthesis-associated factor of the genome, for example, reversible regulatory genes such as the FAD2 gene, FAD3 gene, FAD6 gene, FAD7 gene and/or FAD8 gene, can be cleaved, resulting in knockdown or gene knockout.

As another example, targeted knockdown may be mediated using an enzymatically inactive CRISPR enzyme fused to a transcriptional repressor domain or chromatin-modified protein to alter transcription, e.g., to block, down-regulate, or reduce transcription of the FAD2 gene, the FAD3 gene, the FAD6 gene, FAD7 gene and/or FAD8 gene.

The production of unsaturated fatty acids can be regulated by an artificially manipulated unsaturated fatty acid biosynthesis-associated factor.

A plant organism with an increased or decreased content of a specific unsaturated

- 59 046169 fatty acid or processed product using a plant organism can be obtained using an artificially manipulated factor associated with the biosynthesis of unsaturated fatty acids.

In one illustrative embodiment of the present invention, the manipulable unsaturated fatty acid biosynthesis-associated factor may provide different manipulable unsaturated fatty acid biosynthesis-associated factors in accordance with the constitutional characteristic of the gRNA-CRISPR enzyme complex (e.g., included in target region of a factor associated with the biosynthesis of unsaturated fatty acids or characterized by an adjacent basic PAM sequence).

Hereinafter, although typical examples of CRISPR enzymes and the unsaturated fatty acid biosynthesis-associated gene have been illustrated, they are purely specific examples and thus the present invention is not limited to them.

For example, if the CRISPR enzyme is a SpCas9 protein, then the PAM sequence is 5'-NGG-3' (N represents A, T, G, or C), and the cleavage region of the base sequence (target region) may be a contiguous region from 1 p.o. - 25 bp, for example, 17 bp - 23 p.o. or 21 bp - 23 bp, in the base sequence adjacent to the 5' end and/or 3' end of the 5'-NGG-3' sequence in the target gene.

The present invention may provide an artificially manipulated unsaturated fatty acid biosynthesis-associated factor, such as an artificially manipulated FAD2 gene, a FAD3 gene, a FAD6 gene, a FAD7 gene, and/or a FAD8 gene that is obtained by

a) deletion of one or more nucleotides of a contiguous region of 1 bp. - 25 bp, for example, 17 bp - 23 bp, in the base sequence adjacent to the 5' end and/or 3' end of the sequence 5'-NGG-3' (N represents A, T, C or G),

b) replacing one or more nucleotides of a contiguous 1 bp region. - 25 bp, for example, 17 bp - 23 bp, in a base sequence adjacent to the 5'-end and/or 3'-end of the 5'-NGG-3' sequence, nucleotides different from the nucleotides of the wild type gene,

c) insertion of one or more nucleotides into a contiguous 1 bp region. - 25 bp, for example, 17 bp - 23 bp, in a base sequence adjacent to the 5' end and/or 3' end of the 5-NGG-3' sequence, or

d) a combination of two or more selected from a) to c), in the nucleic acid sequence of an unsaturated fatty acid biosynthesis-associated factor.

For example, if the CRISPR enzyme is a CjCas9 protein, then the PAM sequence is 5'-NNNNRYAC-3' (each N independently represents A, T, C or G, R represents A or G, and Y represents C or T ), and the cleaved base sequence region (target region) may be a contiguous 1 bp region. - 25 bp, for example, 17 bp - 23 p.o. or 21 acting - 23 a.i., in the base sequence adjacent to the 5'-end and/or 3'-end of the sequence 5'-NNNNRYAC-3' in the target gene.

The present invention may provide an artificially manipulated unsaturated fatty acid biosynthesis-associated factor, for example, an artificially manipulated FAD2 gene, FAD3 gene, FAD6 gene, FAD7 gene and/or FAD8 gene, which is obtained by a') deletion of one or more than nucleotides of a continuous section of 1 bp. - 25 bp, for example, 17 bp - 23 bp, in the base sequence adjacent to the 5' end and/or 3' end of the sequence 5'-NNNNRYAC-3' (each N independently represents A, T, C or G, R represents A or G, and Y is C or T), b') substitution of one or more nucleotides of a contiguous 1 bp region. - 25 bp, for example, 17 bp - 23 bp, in a base sequence adjacent to the 5' end and/or 3' end of the sequence 5'-NNNNRYAC-3', nucleotides other than those of the wild type gene, c') insertion of one or more nucleotides into a continuous section of 1 bp. - 25 bp, for example, 17 bp - 23 bp, in a base sequence adjacent to the 5'-end and/or 3'-end of the sequence 5'-NNNNRYAC-3', or d') combination of two or more selected from a') - c' ), in the nucleic acid sequence of the factor associated with the biosynthesis of unsaturated fatty acids.

For example, if the CRISPR enzyme is a StCas9 protein, then the PAM sequence is 5'-NNAGAAW-3' (each N independently represents A, T, C, or G, and W represents A or T), and the cleavage region of the base sequence (target region) may be a contiguous 1 bp region. - 25 bp, for example, 17 bp - 23 p.o. or 21 bp 23 bp, in a base sequence adjacent to the 5' end and/or 3' end of the 5'-NNAGAAW-3' sequence in the target gene.

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The present invention may provide an artificially manipulated unsaturated fatty acid biosynthesis-associated factor, such as an artificially manipulated FAD2 gene, a FAD3 gene, a FAD6 gene, a FAD7 gene, and/or a FAD8 gene that is obtained by

a) deletion of one or more nucleotides of a contiguous region of 1 bp. - 25 bp, for example, 17 bp - 23 bp, in the base sequence adjacent to the 5' end of the sequence 5'-NNAGAAW-3' (each N independently represents A, T, C or G, and W represents A or T),

b) replacing one or more nucleotides of a contiguous 1 bp region. - 25 bp, for example, 17 bp - 23 bp, in a base sequence adjacent to the 5'-end and/or 3'-end of the sequence 5'-NNAGAAW-3', nucleotides different from those of the wild-type gene,

c) insertion of one or more nucleotides into a contiguous 1 bp region. - 25 bp, for example, 17 bp - 23 bp, in a base sequence adjacent to the 5'-end and/or 3'-end of the sequence 5'-NNAGAAW-3', or

d) a combination of two or more selected from a) to c), in the nucleic acid sequence of an unsaturated fatty acid biosynthesis-associated factor.

For example, if the CRISPR enzyme is an NmCas9 protein, then the PAM sequence is 5'-NNNNGATT-3' (each N independently represents A, T, C, or G), and the cleavage region of the base sequence (target region) may represent is a continuous section of 1 bp. - 25 bp, for example, 17 bp - 23 p.o. or 21 bp - 23 bp, in the base sequence adjacent to the 5'-end and/or 3'-end of the sequence 5'-NNNNGATT-3' in the target gene.

The present invention may provide an artificially manipulated unsaturated fatty acid biosynthesis-associated factor, for example, an artificially manipulated FAD2 gene, FAD3 gene, FAD6 gene, FAD7 gene and/or FAD8 gene, which is obtained by a') deletion of one or more than nucleotides of a continuous section of 1 bp. - 25 bp, for example, 17 bp - 23 bp, in the base sequence adjacent to the 5' end and/or 3' end of the sequence 5'-NNNNGATT-3' (each N independently represents A, T, C or G), b') substitution of one or more nucleotides in a contiguous 1 bp region. - 25 bp, for example, 17 bp - 23 bp, in a base sequence adjacent to the 5'-end and/or 3'-end of the sequence 5'-NNNNGATT-3', nucleotides other than those of the wild-type gene, c') insertion of one or more nucleotides into a continuous section of 1 bp. - 25 bp, for example, 17 bp - 23 bp, in the base sequence adjacent to the sequence 5'-NNNNGATT-3', or d') combination of two or more selected from a') - c'), in the nucleic acid sequence associated with the biosynthesis of unsaturated fatty acids acid factor.

For example, if the CRISPR enzyme is the SaCas9 protein, then the PAM sequence is $ -NNGRR(T)-3 (each N independently represents A, T, C, or G, R represents A or G, and (T) represents randomly added sequence), and the cleaved base sequence region (target region) may be a contiguous 1 bp region. - 25 bp, for example, 17 bp - 23 p.o. or 21 bp - 23 bp, in the base sequence adjacent to the 5' end and/or 3' end of the $ -NNGRR(T)-3 sequence in the target gene.

The present invention may provide an artificially manipulated unsaturated fatty acid biosynthesis-associated factor, such as an artificially manipulated FAD2 gene, a FAD3 gene, a FAD6 gene, a FAD7 gene, and/or a FAD8 gene that is obtained by a) deleting one or more nucleotides of a continuous section of 1 bp. - 25 bp, for example, 17 bp - 23 bp, in the base sequence adjacent to the 5' end and/or 3' end of the sequence 5 -NNGRR(T)-3 (each N independently represents A, T, C or G, R represents A or G, and (T) is a randomly added sequence), b') substitution of one or more nucleotides of a contiguous region of 1 a.a. - 25 i.o., for example, 17 p.o. - 23 bp, in a base sequence adjacent to the 5'-end and/or 3'-end of the sequence 5'-NNGRR(T)-3', nucleotides different from such nucleotides of the wild-type gene,

c) insertion of one or more nucleotides into a contiguous 1 bp region. - 25 bp, for example, 17 bp - 23 bp, in a base sequence adjacent to the sequence $ -NNGRR(T)-3, or

d) a combination of two or more selected from a) to c), in the nucleic acid sequence of an unsaturated fatty acid biosynthesis-associated factor.

For example, if the CRISPR enzyme is the Cpf1 protein, then the PAM sequence is $-TTN-3 (N represents A, T, C, or G), and the cleaved base sequence region (target region) may be a contiguous 10 bp region .O. - 30 bp, for example, 15 bp -26 bp, 17 bp - 30 p.o. or 17 bp - 26 bp, in a base sequence adjacent to the 5' end or 3' end of the 5'-TTN-3' sequence.

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The Cpfl protein can be obtained from a microorganism such as

Parcubacteria bacterium (GWC2011_GWC2_44_17), Lachnospiraceae bacterium (MC2017), Butyrivibrio proteoclasiicus, Peregrinibacteria bacterium (GW2011_GWA_33_10), Acidaminococcus sp. (BV3L6), Porphyromonas macacae, Lachnospiraceae bacterium (ND2006), Porphyromonas crevioricanis, Prevotella disiens, Moraxella bovoculi (237), Smiihella sp. (SC KO8D17), Leptospira inadai, Lachnospiraceae bacterium (MA2020), Francisella novicida (UI 12), Candidatus Methanoplasma termitum or Eubacterium eligens, e.g. Parcubacteria bacterium (GWC2011_GWC2_44_17), Peregrinibacterium bacterium (GW2011_GWA_33_10), Acidaminococcus sp . (BV3L6) the present invention is not limited to them.

The present invention may provide an artificially manipulated unsaturated fatty acid biosynthesis-associated factor, such as an artificially manipulated FAD2 gene, a FAD3 gene, a FAD6 gene, a FAD7 gene, and/or a FAD8 gene that is obtained by

a.....) deletions of one or more nucleotides of a contiguous section of 10 bp. - 30 bp, for example 15

By. - 26 bp, in the base sequence adjacent to the 5' end and/or 3' end of the sequence 5'-TTN-3' (N represents A, T, C or G),

b.....) substitution of one or more nucleotides of a contiguous 10 bp region. - 30 bp, for example 15

By. - 26 bp, in the base sequence adjacent to the 5' end and/or 3' end of the sequence

S'-TTN-3' „„ „ „ „ „ „ „ __ „ „ ____ n ¹¹ n n nucleotides other than those of the wild-type gene,

c.....) insertion of one or more nucleotides in a contiguous 10 bp region. - 30 bp, for example 15

By. - 26 bp, in a base sequence adjacent to the 5' end and/or 3' end of the 5'-TTN-3' sequence, or

d.....) combinations of two or more selected from a.....) - c.....), in the nucleic acid sequence of the factor associated with the biosynthesis of unsaturated fatty acids.

In another exemplary embodiment, if the unsaturated fatty acid biosynthesis-associated factor is a protein, the manipulated protein includes all proteins involved in driving the new or modified unsaturated fatty acid biosynthesis through direct or indirect action of the gRNA-CRISPR enzyme complex.

For example, the manipulated protein may be a protein expressed by an unsaturated fatty acid biosynthesis-associated factor (gene) that has been manipulated by a gRNA-CRISPR enzyme complex or another protein that is increased or decreased by such protein activity when the present invention is not limited to them.

The manipulated unsaturated fatty acid biosynthesis-associated factor (protein) may have an amino acid composition and activity corresponding to the composition of the manipulated unsaturated fatty acid biosynthesis-associated factor (gene).

One embodiment may include (i) an artificially manipulated protein whose expression characteristics are altered.

For example, a protein modification may have one or more of the following characteristics:

a decrease or increase in the level of expression in accordance with the deletion or insertion of one or more nucleotides in a contiguous region of 1 bp. - 50 p.o., 1 p.o. - 40 p.o., 1 p.o. - 30 p.o. and preferably 3 bp. - 25 p.o. in the base sequence of the PAM sequence in the nucleic acid sequence of the unsaturated fatty acid biosynthesis-associated factor or adjacent to its 5' end and/or 3' end;

decreasing or increasing the level of expression in accordance with the substitution of one or more nucleotides other than those of the wild type gene;

decreasing or increasing the level of expression, expression of a fusion protein, or independent expression of a specific protein in accordance with the insertion of one or more foreign nucleotides; and decreasing or increasing the expression level of the third protein under the influence of the expression characteristics of the above-described proteins.

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Can be represented by (iii) an artificially manipulated protein whose structural characteristics are changed.

For example, a protein modification may have one or more of the following characteristics:

a change in codons, amino acids, and three-dimensional structure due to the deletion or insertion of one or more nucleotides in a contiguous 1-bp region. -50 bp, 1 bp - 40 p.o., 1 p.o. - 30 p.o. and preferably 3 bp. - 25 p.o. in the base sequence of the PAM sequence in the nucleic acid sequence of the unsaturated fatty acid biosynthesis-associated factor or adjacent to its 5' end and/or 3' end;

a change in codons, amino acids and thereby in three-dimensional structure in accordance with the substitution of one or more nucleotides other than those of the wild type gene;

a change in the codons, amino acids and three-dimensional structure of either a structure fused to a specific protein or an independent structure from which a specific protein is isolated, in accordance with the insertion of one or more foreign nucleotides; and a change in the codons, amino acids and three-dimensional structure of the third protein under the influence of the above-described protein with an altered structural characteristic.

Can be represented by (iii) an artificially manipulated protein whose functional characteristics are changed.

For example, a protein modification may have one or more of the following characteristics:

activation or inactivation of a specific function due to protein modification caused by the deletion or insertion of one or more nucleotides in a contiguous 1 bp region. - 50 p.o., 1 p.o. - 40 p.o., 1 p.o. - 30 p.o. and preferably 3 bp. - 25 p.o. in the base sequence of the PAM sequence in the nucleic acid sequence of the unsaturated fatty acid biosynthesis-associated factor or adjacent to its 5' end and/or 3' end;

activation or inactivation of a specific function or introduction of a new function due to protein modification caused by the substitution of one or more nucleotides other than those of the wild type gene;

activation or inactivation of a specific function or introduction of a new function due to protein modification caused by the insertion of one or more foreign nucleotides, in particular, the introduction of a third function in addition to an existing function due to fusion or independent expression of a specific protein; and a change in the function of the third protein under the influence of the above-described protein with altered functional characteristics.

In addition, a protein that has been artificially manipulated by chemical modification of one or more nucleotides in the nucleic acid sequence of which the unsaturated fatty acid biosynthesis-associated factor is composed may be included.

For example, one or more of the protein expression, structural and functional characteristics caused by methylation, acetylation, phosphorylation, ubiquitinylation, ADP-ribosylation, myristylation and glycosylation may be altered.

For example, a third structure and function may be obtained by linking a third protein to the nucleic acid sequence of a gene through chemical modification of the nucleotides.

5. Other additional components.

An additional component may be selectively added to enhance the efficiency of the guide nucleic acid-editing protein complex or to improve the efficiency of repair of a damaged gene or nucleic acid.

The additional component can be selectively used to improve the efficiency of the guide nucleic acid-editing protein complex.

Activator.

The additional component can be used as an activator to increase the efficiency of cleavage of the target nucleic acid, gene or chromosome of the guide nucleic acid-editing protein complex.

The term activator refers to a nucleic acid that serves to stabilize the association between a guide nucleic acid-editing protein complex and a target nucleic acid, gene or chromosome or to allow the guide nucleic acid-editing protein complex to more easily reach a target nucleic acid, gene or chromosome.

The activator may be a double-stranded nucleic acid or a single-stranded nucleic acid.

The activator can be linear or ring.

An activator can be divided into a helper, which stabilizes the association between the guide nucleic acid-editing protein complex and the target nucleic acid, gene or chromosome, and a chaperone, which serves to allow the guide nucleic acid-editing protein complex to more easily reach the target nucleic acid, gene or chromosome. chromosomes.

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The helper can increase the efficiency of cleavage of the guide nucleic acid-editing protein complex towards a target nucleic acid, gene or chromosome.

For example, a helper includes a nucleic acid sequence having homology to a target nucleic acid, gene or chromosome. Therefore, if the guide nucleic acid-editing protein complex is associated with a target nucleic acid, gene or chromosome, then a homologous nucleic acid sequence included in the helper can form an additional complementary bond with the target nucleic acid, gene or chromosome to stabilize the connection between the guide nucleic acid complex editing protein and target nucleic acid, genome or chromosome.

The chaperone can increase the efficiency of cleavage of the guide nucleic acid-editing protein complex towards a target nucleic acid, gene or chromosome.

For example, a chaperone includes a nucleic acid sequence having homology to the target nucleic acid, gene or chromosome. In this case, a homologous nucleic acid sequence included in the chaperone may partially form a complementary bond with the guide nucleic acid of the guide nucleic acid-editing protein complex. Therefore, a guide that partially forms a complementary bond with the guide nucleic acid-editing protein complex can partially form a complementary bond with the target nucleic acid, gene, or chromosome and, as a result, can ensure that the guide nucleic acid-editing protein complex accurately reaches the location of the target nucleic acid, gene. or chromosomes.

A homologous nucleic acid sequence may have at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or greater homology or complete homology.

In addition, an additional component can be selectively used to improve the efficiency of repair of damaged genes or nucleic acids.

Assistant.

An additional component can be used as an assistant to improve the efficiency of repair of damaged genes or nucleic acids.

The term helper refers to a nucleic acid that participates in the repair process or enhances the efficiency of repair of a damaged gene or nucleic acid, for example, a gene or nucleic acid cleaved by a guide nucleic acid-editing protein complex.

The helper may be a double-stranded nucleic acid or a single-stranded nucleic acid.

The assistant may be present in a linear or circular form.

The helper can be divided into an NHEJ helper, which participates in the repair process using NHEJ or improves the repair efficiency, and an HDR helper, which participates in the repair process using HDR or improves the repair efficiency according to the repair method.

The NHEJ helper may participate in the repair process or improve the efficiency of repair of a damaged gene or nucleic acid using NHEJ.

For example, the NHEJ helper may include a nucleic acid sequence that has homology to a portion of the damaged nucleic acid sequence. Here, the homologous nucleic acid sequence may include a nucleic acid sequence having homology with a nucleic acid sequence at one end (for example, the 3' end) of the damaged nucleic acid sequence, and include a nucleic acid sequence having homology with a nucleic acid sequence at the other end (eg, 5' end) of a damaged nucleic acid sequence. In addition, a nucleic acid sequence having homology to each of the base sequences in the 3'-5' direction and in the 5'-3' direction of the damaged nucleic acid sequence may be included. A nucleic acid sequence having such homology can help place two parts of a damaged nucleic acid sequence in close proximity, thereby increasing the efficiency of repair of the damaged nucleic acid by NHEJ.

An HDR helper can participate in the repair process or improve the efficiency of repair of a damaged gene or nucleic acid using HDR.

For example, the HDR helper may include a nucleic acid sequence that has homology to a portion of the damaged nucleic acid sequence. Here, the homologous nucleic acid sequence may include a nucleic acid sequence having homology with a nucleic acid sequence at one end (for example, the 3' end) of the damaged nucleic acid sequence, and a nucleic acid sequence having homology with a nucleic acid sequence at the other

- 64 046169 end (eg 5' end) of the damaged nucleic acid sequence. Alternatively, a nucleic acid sequence having homology to each of the base sequences in the 3'-5' direction and in the 5'-3' direction of the damaged nucleic acid sequence may be included. A nucleic acid sequence having such homology can serve as a template for a damaged nucleic acid sequence to improve the efficiency of repair of the damaged nucleic acid using HDR.

As another example, the HDR assistant may include a nucleic acid sequence having homology to a portion of the damaged nucleic acid sequence and a specific nucleic acid, such as a nucleic acid or gene, to be inserted. Here, the homologous nucleic acid sequence may include a nucleic acid sequence having homology with each of the base sequences in the 3'-5' direction and in the 5'-3' direction of the damaged nucleic acid sequence. The specific nucleic acid may be located between a nucleic acid sequence having homology to a base sequence in the 5'-3' direction of the damaged nucleic acid, and a nucleic acid sequence having homology to the base sequence in the 3'-5' direction of the damaged nucleic acid. The nucleic acid sequence having such homology and the specific nucleic acid can serve as a donor for insertion of the specific nucleic acid into the damaged nucleic acid, thereby increasing the efficiency of HDR against the knockin.

A homologous nucleic acid sequence may have at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or greater homology or complete homology.

6. Subject.

The term subject refers to an organism into which a guide nucleic acid, editing protein, or guide nucleic acid-editing protein complex is introduced, an organism in which the guide nucleic acid, editing protein, or guide nucleic acid-editing protein complex functions, or a sample or specimen. obtained from the body.

The subject may be an organism containing a target nucleic acid, gene, chromosome, or guide nucleic acid-editing protein complex protein.

An organism may be a cell, a tissue, or a plant. The cells may be eukaryotic cells. Eukaryotic cells may be plant cells.

The tissue may be plant tissue such as a leaf, stem, root, flower, fruit or callus, etc., etc.

A plant can be a plant at various stages from seed to mature organism.

The plant may be a plant organism containing unsaturated fatty acids.

In addition, the subject may be a probe or specimen containing a target nucleic acid, gene, chromosome, or protein of the guide nucleic acid-editing protein complex.

The sample or specimen may be obtained from a plant organism.

In accordance with the present invention, as a specific example, the subject may contain a target gene or nucleic acid of the guide nucleic acid-editing protein complex.

In this case, the target gene may be an unsaturated fatty acid biosynthesis-associated factor, for example, the FAD2 gene, the FAD3 gene, the FAD6 gene, the FAD7 gene and/or the FAD8 gene.

The target gene may be a wild type gene or a modified form in the wild type.

In one illustrative embodiment of the present invention, the subject may contain a gene or nucleic acid manipulated by a guide nucleic acid-editing protein complex.

In this case, the manipulated gene may be an unsaturated fatty acid biosynthesis-associated factor, for example, the FAD2 gene, the FAD3 gene, the FAD6 gene, the FAD7 gene and/or the FAD8 gene.

In this case, the targeting nucleic acid may target an unsaturated fatty acid biosynthesis-associated factor, for example, the FAD2 gene, the FAD3 gene, the FAD6 gene, the FAD7 gene, and/or the FAD8 gene.

The guide nucleic acid may be a nucleic acid sequence complementary to the target sequence of the FAD2 gene, FAD3 gene, FAD6 gene, FAD7 gene and/or FAD8 gene.

The guide nucleic acid can target one or more genes.

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The guide nucleic acid can simultaneously target two or more genes. In this case, the two or more genes may be homologous or heterologous genes.

The guide nucleic acid can target one or more target sequences.

The guide nucleic acid can be designed in different forms according to the number or locations of target sequences.

In one illustrative embodiment of the present invention, the targeting nucleic acid may be a nucleic acid sequence complementary to one or more target sequences of the sequences listed in table. 1.

In a specific embodiment, a guide nucleic acid sequence corresponding to any of the target sequences of SEQ ID NO: 1-30 is provided for artificial manipulation of the FAD2 gene.

In a certain embodiment, for artificial manipulation of the FAD2 gene, an editing protein is provided that interacts with a nucleic acid guide sequence corresponding, for example, to form a complex with any of the target sequences of SEQ ID NO: 1-30, for example, SEQ ID NO: 7 or thirty.

In a certain embodiment, a nucleic acid modification product of each gene that artificially manipulates a region of the target sequence under any of SEQ ID NO: 1-30, for example, SEQ ID NO: 7 or 30, and an expression product thereof are provided.

7. Delivery.

The guide nucleic acid, editing protein, or guide nucleic acid-editing protein complex can be delivered to or introduced into a subject using various delivery methods and various forms.

The guide nucleic acid may be delivered or introduced into the subject in the form of DNA, RNA, or a mixed form.

The editing protein may be delivered to or introduced into a subject in the form of DNA, RNA, a mixture of DNA/RNA, a peptide, a polypeptide, each encoding an editing protein, or in the form of a protein.

The guide nucleic acid-editing protein complex may be delivered to or introduced into the target in the form of DNA, RNA, or a combination thereof that encodes each component, that is, the guide nucleic acid or editing protein.

The guide nucleic acid-editing protein complex may be delivered to or administered to a subject as a complex of a guide nucleic acid in the form of DNA, RNA, or a mixture thereof, and an editing protein in the form of a peptide, polypeptide, or protein.

In addition, an additional component capable of enhancing or inhibiting the effectiveness of the guide nucleic acid-editing protein complex may be delivered to or administered to a subject by various delivery methods and in various forms.

The additional component may be delivered to or administered to the subject in the form of DNA, RNA, a DNA/RNA mixture, peptide, polypeptide or protein.

i) Delivery in the form of DNA, RNA or a mixture of both.

The guide nucleic acid and/or editing protein is encoded in the form of DNA, RNA, or a mixture thereof, which can be delivered to or introduced into a subject using a method known in the art.

Or, the guide nucleic acid and/or editing protein is encoded in the form of DNA, RNA, or a mixture thereof, which can be delivered to or introduced into a subject using a vector, without using a vector, or a combination thereof.

The vector may be a viral vector or a non-viral vector (eg, a plasmid).

The non-vector form may be naked DNA, complex DNA, or mRNA.

Vector based introduction.

A nucleic acid sequence encoding a targeting nucleic acid and/or an editing protein may be delivered to or introduced into a subject using a vector.

The vector may include a nucleic acid sequence encoding a guide nucleic acid and/or an editing protein.

For example, the vector may simultaneously include nucleic acid sequences that respectively encode a guide nucleic acid and an editing protein.

For example, the vector may include a nucleic acid sequence encoding a targeting nucleic acid.

As an example, the domains included in the guide nucleic acid may be contained all together in a single vector, or they may be separated and then inserted into different vectors.

For example, the vector may include a nucleic acid sequence encoding an edit

- 66 046169 tyrating protein.

In one example, in the case of an editing protein, the nucleic acid sequences encoding the editing protein may be contained in a single vector, or they may be separated and then included in multiple vectors.

The vector may include one or more regulatory/control components.

In this case, the regulatory/control components may include a promoter, an enhancer, an intron, a polyadenylation signal, a Kozak consensus sequence, an internal ribosome entry site (IRES), a splice acceptor, and/or a 2A sequence.

The promoter may be a promoter recognized by RNA polymerase II. The promoter may be a promoter recognized by RNA polymerase III. The promoter may be an inducible promoter. The promoter may be a subject-specific promoter. The promoter may be a viral or non-viral promoter.

In the case of a promoter, a suitable promoter may be used in accordance with a control region (ie, a nucleic acid sequence encoding a guide nucleic acid or editing protein).

For example, a promoter useful for the guide nucleic acid may be an H1, EF-1a, tRNA, or U6 promoter. For example, a promoter useful for an editing protein may be a CMV, EF-1a, EFS, MSCV, PGK, or CAG promoter.

For example, a promoter useful for a nucleic acid guide and/or editing protein may be a root-specific expression promoter, a seed-specific promoter, a whole-body inducible expression promoter, or a leaf or other tissue-specific promoter.

The vector may be a viral vector or a recombinant viral vector.

The virus may be a DNA virus or an RNA virus.

Here, the DNA virus may be a double-stranded DNA virus (dsDNA) or a single-stranded DNA virus (ssDNA).

In this case, the RNA virus may be a single-stranded RNA (ssRNA) virus.

The virus may be a mosaic virus, a retrovirus, a lentivirus, an adenovirus, an adeno-associated virus (AAV), a vaccinia virus, a poxvirus, or a herpes simplex virus, but the present invention is not limited to them.

Typically, a virus can infect a host (eg, a cell) thereby introducing a nucleic acid encoding the genetic information of the virus into the host, or inserting a nucleic acid encoding the genetic information into the genome of the host. The guide nucleic acid and/or editing protein can be introduced into a subject using a virus having such a characteristic. The guide nucleic acid and/or editing protein introduced using a virus can be transiently expressed in a subject (eg, cells). Alternatively, the targeting nucleic acid and/or editing protein introduced using the virus may be continuously expressed in a subject (eg, cells) over an extended period of time (eg, 1, 2 or 3 weeks, 1, 2, 3, 6 or 9 months, 1 or 2 years or permanently).

The packaging capacity of the virus can vary from at least 2 kb. up to 50 tp. according to the type of virus. Depending on such packaging ability, a viral vector including a targeting nucleic acid or an editing protein, or a viral vector including both a targeting nucleic acid and an editing protein, can be constructed. Alternatively, a viral vector can be constructed including a guide nucleic acid, an editing protein, and additional components.

In one example, a nucleic acid sequence encoding a guide nucleic acid and/or an editing protein can be delivered or introduced using a recombinant mosaic virus.

As another example, a nucleic acid sequence encoding a guide nucleic acid and/or an editing protein can be delivered or introduced using a recombinant adenovirus.

As a further example, a nucleic acid sequence encoding a guide nucleic acid and/or an editing protein can be delivered or introduced by recombinant AAV.

As another example, a nucleic acid sequence encoding a guide nucleic acid and/or an editing protein may be delivered or introduced by a hybrid virus, such as one or more of the virus hybrids provided herein.

In addition, the vector can be incorporated into a bacterium and introduced into a subject.

In this case, the vector can be included in Agrobacterium and introduced into the subject, but the present invention is not limited to this.

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Generally, the most widely used method of transferring the required genetic material into a subject is using Agrobacteria, and in the case of a plant, to genetically modify the plant, the Agrobacterium DNA can be inserted into the chromosome of the plant organism in a nucleic acid-protein form called a plasmid. It can serve to transfer genetic material into the cells of a plant organism, while the transferred genetic material fuses in the cells. The above method can be widely used for producing a genetically modified Agrobacterium culture, and, in addition, it can also be used as a system for studying the response of cells to genetic transformation.

Introduction other than vector.

The nucleic acid sequence encoding the targeting nucleic acid and/or editing protein may be delivered to or introduced into the subject using a non-vector form.

The non-vector form may include a nucleic acid sequence encoding a guide nucleic acid and/or an editing protein.

The non-vector form may be naked DNA, complex DNA, mRNA, or a combination thereof.

The non-vector form may be delivered to or introduced into the subject by electroporation, particle bombardment, sonication, magnetofection, transient compression, or cell squeeze (e.g., as described in the literature [Lee, et al. (2012) Nano Lett ., 12, 6322-6327]), lipid-mediated transfection, dendrimer, nanoparticles, calcium phosphate, silica, silicate (Ormosil) or a combination thereof.

By way of example, delivery by electroporation may be accomplished by mixing cells and a nucleic acid sequence encoding a targeting nucleic acid and/or editing protein in a cartridge, chamber, or cuvette and applying an electrical stimulus of a predetermined duration and amplitude to the cells.

As another example, a non-vector form can be delivered using nanoparticles. Nanoparticles can be inorganic nanoparticles (eg, magnetic nanoparticles, silica nanoparticles, etc.) or organic nanoparticles (eg, polyethylene glycol (PEG)-coated lipid, etc.). The outer surface of the nanoparticles can be conjugated to a positively charged polymer that is capable of attachment (e.g., polyethylenimine, polylysine, polyserine, etc.).

In a certain embodiment, the non-vector form can be delivered using a lipid shell.

In a certain embodiment, the non-vector form can be delivered using an exosome. An exosome is an endogenous protein and RNA transport nanovesicle that can deliver RNA to the brain and other target organ.

In a certain embodiment, the non-vector form can be delivered using a liposome. A liposome is a spherical vesicular structure that consists of a single or multilayer lamellar lipid bilayer surrounding an internal aqueous compartment and an outer lipophilic phospholipid bilayer that is relatively opaque. Although a liposome can be made from several different types of lipids, phospholipids are the most widely used to prepare liposomes as drug carriers.

Other additives may be included.

ii) Delivery in the form of a peptide, polypeptide or protein.

The editing protein, in the form of a peptide, polypeptide or protein, can be delivered to or introduced into a subject using a method known in the art.

The peptide, polypeptide, or protein forms may be delivered to or introduced into a subject by electroporation, microinjection, transient compression, or cell compression (e.g., as described in the literature [Lee, et al. (2012) Nano Lett., 12, 6322-6327 ]), lipid-mediated transfection, nanoparticles, liposomes, peptide-mediated delivery, or combinations thereof.

The peptide, polypeptide or protein can be delivered with a nucleic acid sequence encoding a guide nucleic acid.

In one example, transfer by electroporation can be accomplished by mixing the cells to which the editing protein will be introduced, with or without a targeting nucleic acid, in a cartridge, chamber, or cuvette and by applying an electrical stimulus of a specified duration and amplitude to the cells.

iii) Delivery in the form of a nucleic acid-protein mixture.

The guide nucleic acid and editing protein can be delivered to or introduced into the subject in the form of a guide nucleic acid-editing protein complex.

For example, the guide nucleic acid may be DNA, RNA, or a combination thereof. The editing protein may be a peptide, polypeptide or protein.

- 68 046169

In one example, the guide nucleic acid and editing protein may be delivered to or introduced into a subject in the form of a guide nucleic acid-editing protein complex comprising a guide nucleic acid such as RNA and an editing protein such as a ribonucleoprotein (RNP).

In accordance with the present invention, as an embodiment of a method for delivering a guide nucleic acid and/or editing protein to a subject, delivery of a gRNA, a CRISPR enzyme, or a gRNA-CRISPR enzyme complex will be described below.

In one embodiment of the present invention, a nucleic acid sequence encoding a gRNA and/or a CRISPR enzyme will be delivered to or introduced into a subject using a vector.

The vector may include a nucleic acid sequence encoding a gRNA and/or a CRISPR enzyme.

For example, the vector may simultaneously include nucleic acid sequences encoding a gRNA and a CRISPR enzyme.

For example, the vector may include a nucleic acid sequence encoding a gRNA.

In one example, the domains contained in the gRNA may be contained in a single vector, or they may be separated and then inserted into different vectors.

For example, the vector may include a nucleic acid sequence encoding a CRISPR enzyme.

In one example, in the case of a CRISPR enzyme, the nucleic acid sequences encoding the CRISPR enzyme may be contained in a single vector, or they may be separated and then inserted into multiple vectors.

The vector may include one or more regulatory/control components.

In this case, regulatory/control components may include a promoter, enhancer, intron, polyadenylation signal, consensus sequence.

Kozak, internal ribosome entry site (IRES), splice acceptor and/or 2A sequence.

The promoter may be a promoter recognized by RNA polymerase II. The promoter may be a promoter recognized by RNA polymerase III. The promoter may be an inducible promoter. The promoter may be a subject-specific promoter. The promoter may be a viral or non-viral promoter.

In the case of a promoter, a suitable promoter may be used in accordance with the control region (ie the nucleic acid sequence encoding the gRNA and/or the CRISPR enzyme).

For example, a promoter useful for gRNA may be an H1, EF-1a, tRNA or U6 promoter. For example, a promoter useful for a CRISPR enzyme may be a CMV, EF-1a, EFS, MSCV, PGK, or CAG promoter.

For example, a promoter useful for a nucleic acid guide and/or editing protein may be a root-specific expression promoter, a seed-specific promoter, a whole-body inducible expression promoter, or a leaf or other tissue-specific promoter.

The vector may be a viral vector or a recombinant viral vector.

The virus may be a DNA virus or an RNA virus.

Here, the DNA virus may be a double-stranded DNA virus (dsDNA) or a single-stranded DNA virus (ssDNA).

In this case, the RNA virus may be a single-stranded RNA (ssRNA) virus.

The virus may be a mosaic virus, a retrovirus, a lentivirus, an adenovirus, an adeno-associated virus (AAV), a vaccinia virus, a poxvirus, or a herpes simplex virus, but the present invention is not limited to them.

Typically, a virus can infect a host (eg, a cell) thereby introducing a nucleic acid encoding the genetic information of the virus into the host, or inserting a nucleic acid encoding the genetic information into the genome of the host. The gRNA and/or CRISPR enzyme can be introduced into a subject using a virus having such characteristics. The gRNA and/or CRISPR enzyme introduced using a virus can be transiently expressed in a subject (eg, cells). Alternatively, the gRNA and/or CRISPR enzyme introduced using a virus may be continuously expressed in a subject (e.g., cells) over an extended period of time (e.g., 1, 2, or 3 weeks, 1, 2, 3, 6, or 9 months, 1 or 2 years or permanently).

The packaging capacity of the virus can vary from at least 2 kb. up to 50 tp. according to the type of virus. Depending on this packaging ability, a viral vector including only gRNA or the CRISPR enzyme, or a viral vector including both

- 69 046169 gRNA, and the CRISPR enzyme. Alternatively, a viral vector can be constructed that includes the gRNA, the CRISPR enzyme, and additional components.

In one example, a nucleic acid sequence encoding a gRNA and/or a CRISPR enzyme can be delivered or introduced using a recombinant mosaic virus.

As another example, a nucleic acid sequence encoding a gRNA and/or a CRISPR enzyme can be delivered or introduced using a recombinant adenovirus.

As a further example, a nucleic acid sequence encoding a gRNA and/or a CRISPR enzyme can be delivered or introduced using recombinant AAV.

As another example, a nucleic acid sequence encoding a gRNA and/or a CRISPR enzyme can be delivered or introduced using one or more hybrid virus hybrids, such as the viruses described herein.

The vector can be incorporated into a bacterium and introduced into a subject.

In this case, the vector can be included in Agrobacterium and introduced into the subject, but the present invention is not limited to this.

As an example, a vector comprising a nucleic acid sequence(s) encoding a gRNA and/or a CRISPR enzyme can be incorporated into Agrobacterium and introduced into a plant organism.

In one illustrative embodiment of the present invention, the gRNA CRISPR enzyme complex can be delivered to or introduced into a subject.

For example, gRNA may be present in the form of DNA, RNA, or a combination thereof. The CRISPR enzyme may be present in the form of a peptide, polypeptide or protein.

In one example, the gRNA and the CRISPR enzyme may be delivered to or introduced into a subject in the form of a gRNA-CRISPR enzyme complex comprising a gRNA-type RNA and a CRISPR-type protein, i.e., a ribonucleoprotein (RNP).

The gRNA-CRISPR enzyme complex can be delivered to or introduced into a subject by electroporation, microinjection, transient compression, or cell compression (e.g., as described in the literature [Lee, et al. (2012) Nano Lett., 12, 6322-6327 ]), lipid-mediated transfection, nanoparticles, liposomes, peptide-mediated delivery, or combinations thereof.

8. Transformant.

The term transformant refers to the organism into which the guide nucleic acid, editing protein, or guide nucleic acid-editing protein complex is introduced, the organism into which the guide nucleic acid, editing protein, or guide nucleic acid-editing protein complex is expressed, or to a sample or specimen obtained from the body.

The transformant may be an organism into which a guide nucleic acid, an editing protein, or a guide nucleic acid-editing protein complex in the form of DNA, RNA, or a combination thereof is introduced.

For example, the transformant may be an organism into which a vector has been introduced comprising a nucleic acid sequence encoding a guide nucleic acid and/or an editing protein. Here, the vector may be a non-viral vector, a viral vector or a recombinant viral vector.

As another example, a transformant may be an organism into which a nucleic acid sequence encoding a guide nucleic acid and/or an editing protein has been introduced in a non-vector form. In this case, the non-vector form may be naked DNA, complex DNA, mRNA, or a combination thereof.

The transformant may be an organism into which a guide nucleic acid, an editing protein, or a guide nucleic acid-editing protein complex in the form of a peptide, polypeptide, or protein has been introduced.

The transformant may be an organism into which a guide nucleic acid, an editing protein, or a guide nucleic acid-editing protein complex has been introduced in the form of DNA, RNA, peptide, polypeptide, protein, or a combination thereof.

For example, the transformant may be an organism into which a guide nucleic acid-editing protein complex has been introduced, comprising an RNA-type guide nucleic acid and a protein-type editing protein.

The transformant may be an organism comprising a target nucleic acid, gene, chromosome, or guide nucleic acid-editing protein complex protein.

An organism may be a cell, tissue, or plant.

The cells may be prokaryotic cells or eukaryotic cells.

Eukaryotic cells may be plant cells, but the present invention is not limited to them.

The tissue may be tissue of a plant organism such as a root, stem, leaf, flower, fruit or callus, etc., etc.

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The transformant may be a plant organism into which a guide nucleic acid, an editing protein, or a guide nucleic acid-editing protein complex has been introduced or expressed, or a probe or sample derived from a plant organism.

The sample or sample may be a root, stem, leaf, flower, fruit, callus or cells thereof.

Application.

One exemplary embodiment of the present invention relates to the use of a composition for artificially manipulating an unsaturated fatty acid biosynthesis-associated factor in a subject, such as a plant, to a plant organism in which the content of a specific unsaturated fatty acid is controlled by an artificially manipulated unsaturated fatty acid biosynthesis-associated factor. manipulation, or to a processed product using it.

Specific unsaturated fatty acids Soybean (Glycine max L.).

Soybean is the most widely cultivated crop in the world and produces vegetable oils and proteins of the highest quality in terms of production and use. Transformation technology is widely used to improve the genetic characteristics of various effective genes in soybean. A transgenic soybean plant organism was constructed using a system for transformation using the Agrobacterium-based cotyledon node (CN) method (Hinchee et al., 1988, Nat. Biotechnol., Vol. 6, 915-922), and recently a system for producing a stable transformant was improved using half-seed explants (Paz et al., 2006, Plant Cell Rep., Vol. 25, 206-213). Moreover, wounding the target site using a mixed use of thiol compound, Agrobacterium concentrate and sonication resulted in a positive improvement in transformation efficiency (Meurer et al., 1998, Plant Cell Rep., Vol. 18, 180-186; Olhoft et al. ., 2003, Planta, Vol. 216, 723-735; Kim et al., 2013, Plant Biotechnol Rep., Vol. 7, 425-433; Kim et al., 2016, Plant Biotechnol Rep., Vol. 10, 257-267).

Soy contains approximately 20% total fat, and the fat is made up of fatty acids. Fatty acids consist of saturated fatty acids and unsaturated fatty acids. Unsaturated fatty acids consist of oleic acid, linoleic acid and α-linolenic acid. Among them, α-linolenic acid is a plant-based omega-3 fatty acid that is known to inhibit the growth of cancer cells, prevent cardiovascular disease, inhibit inflammation and blood clotting, and break down fat. Linoleic acid is an omega-6 fatty acid that is known to stimulate cancer cell growth, lower blood pressure, cause inflammation and thrombosis, and promote fat storage in a way that α-linolenic acid does not.

A low omega-6/omega-3 fat ratio has been reported to inhibit the development of the above-mentioned diseases. In some examples, if the ratio is 4:1, then the prevention of cardiovascular disease can be excellent, and blood circulation can be improved if the ratio is 2-3:1, and inflammation in rheumatoid arthritis can be inhibited, while it is known that the ideal ratio is 2-1:1. Soybean oil contains 54% omega-6 fatty acids and 8% omega-3 fatty acids, and the ratio of the two fatty acids is a very high 6-7:1.

In soybean unsaturated fatty acid metabolism, it was known that the FAD2 gene serves to convert oleic acid to linoleic acid, and the FAD3 gene serves to convert linoleic acid to α-linolenic acid. Alterations in oleic and linoleic acids in fatty acid metabolism have been reported as a result of mutation of the FAD2 gene. When a mutation occurs in the FAD2 gene, the oleic acid content increases and the linoleic acid content decreases, so the ratio of linoleic acid to α-linolenic acid is adjusted, but there is a limitation in determining a satisfactory ratio of 4:1 or less. Therefore, it is necessary to control the content of oleic acid and linoleic acid.

Specific unsaturated fatty acids.

In one exemplary embodiment of the present invention, a plant organism enriched in a specific unsaturated fatty acid or a processed product thereof may be provided.

In this case, the specific unsaturated fatty acid may be a C824:D1 unsaturated fatty acid.

The specific unsaturated fatty acid may be a C16-22:D1 unsaturated fatty acid.

The specific unsaturated fatty acid may be a C18:D1 unsaturated fatty acid.

The specific unsaturated fatty acid may be oleic acid.

Alternatively, the specific unsaturated fatty acid may be an unsaturated fatty acid obtained by removing one double bond from the C8-24:D2 unsaturated fatty acid.

- 71 046169 saturated fatty acid, the specific unsaturated fatty acid may be an unsaturated fatty acid obtained by removing one double bond from a C16-22:D2 unsaturated fatty acid, the specific unsaturated fatty acid may be an unsaturated fatty acid obtained by removing one double bond bonds from a C18:D2 unsaturated fatty acid, and the specific unsaturated fatty acid may be an unsaturated fatty acid obtained by removing one double bond from linoleic acid.

Another exemplary embodiment of the present invention relates to a plant organism with a reduced content of a specific unsaturated fatty acid or to a processed product obtained using it.

In this case, the specific unsaturated fatty acid may be a C824:D2 unsaturated fatty acid.

The specific unsaturated fatty acid may be a C16-22:D2 unsaturated fatty acid.

The specific unsaturated fatty acid may be a C18:D2 unsaturated fatty acid.

The specific unsaturated fatty acid may be linoleic acid.

Alternatively, the specific unsaturated fatty acid may be an unsaturated fatty acid obtained by forming a single double bond in a C8-24:D1 unsaturated fatty acid, the specific unsaturated fatty acid may be an unsaturated fatty acid obtained by forming a single double bond in a C16- 22:D1 unsaturated fatty acid, the specific unsaturated fatty acid may be an unsaturated fatty acid obtained by forming one double bond in the C18:D1 unsaturated fatty acid, and the specific unsaturated fatty acid may be an unsaturated fatty acid obtained by forming one double bond in oleic acid.

Alternatively, the specific unsaturated fatty acid may be an unsaturated fatty acid obtained by removing one double bond from a C8-24:D3 unsaturated fatty acid, the specific unsaturated fatty acid may be an unsaturated fatty acid obtained by removing one double bond from a C16- 22:D3 unsaturated fatty acid, the specific unsaturated fatty acid may be an unsaturated fatty acid obtained by removing one double bond from a C18:D3 unsaturated fatty acid, and the specific unsaturated fatty acid may be an unsaturated fatty acid obtained by removing one double bond from α-linolenic acid.

In one embodiment, the specific unsaturated fatty acid may be a C18:D1 unsaturated fatty acid or a C18:D2 unsaturated fatty acid.

In one embodiment, the specific unsaturated fatty acid may be oleic acid or linoleic acid.

In another illustrative embodiment, the present invention may relate to the use of a system for controlling an additional third mechanism in the body that is involved in various functions of a specific factor whose function has been artificially modified (eg, a gene known as unsaturated fatty acid biosynthesis-associated factor).

For example, the specific factor whose function is artificially modified may be one or more genes selected from the FAD2 gene, the FAD3 gene, the FAD6 gene, the FAD7 gene, and the FAD8 gene.

The third mechanism may be a plant mechanism other than unsaturated fatty acid biosynthesis involved in these genes.

Composition intended for control of unsaturated fatty acid.

One exemplary embodiment of the present invention relates to a composition used to control the unsaturated fatty acid content of a plant using an artificially manipulated unsaturated fatty acid biosynthesis-associated factor.

The composition may comprise an artificially manipulable unsaturated fatty acid biosynthesis-associated factor, or a manipulable composition that is capable of artificially manipulating an unsaturated fatty acid biosynthesis-associated factor.

In one illustrative embodiment, the composition may contain an unsaturated fatty acid biosynthesis-associated factor, that is, a gene and/or protein.

In one illustrative embodiment, the composition may include a manipulable composition that may artificially manipulate associated

- 72 046169 bath with biosynthesis of unsaturated fatty acids factor.

The composition intended for manipulation may contain a guide nucleic acid-editing protein complex.

The composition to be manipulated may include a guide nucleic acid and/or an editing protein.

The composition to be manipulated may include a nucleic acid encoding a guide nucleic acid and/or an editing protein.

The composition to be manipulated may include a virus containing a nucleic acid encoding a guide nucleic acid and/or an editing protein.

In another illustrative embodiment, the composition may further include an additional element.

The additional factor may include a suitable carrier for its transfer into the plant body of the subject.

In an illustrative embodiment, the composition may include an expression product of an unsaturated fatty acid biosynthesis-associated factor that is manipulated to an amount sufficient to increase or decrease the content of the specific unsaturated fatty acid.

The term "amount sufficient to increase or decrease the content of a specific unsaturated fatty acid" means an effective amount necessary to increase or decrease the content of a specific unsaturated fatty acid.

In one illustrative embodiment, the present invention may relate to the following compositions for the control of unsaturated fatty acid:

to a composition intended to control the content of a specific unsaturated fatty acid, which includes a targeting nucleic acid capable of forming a complementary bond independently with one or more target sequences in the nucleic acid sequence of one or more genes selected from the group consisting of a FAD2 gene, a FAD3 gene, the FAD6 gene, the FAD7 gene and the FAD8 gene, or a nucleic acid sequence encoding it, and to an editing protein or a nucleic acid sequence encoding it;

to a composition intended for controlling the content of a specific unsaturated fatty acid, which includes a guide nucleic acid capable of forming a complementary bond independently with the target sequence of one or more genes selected from the group consisting of the FAD2 gene, the FAD3 gene, the FAD6 gene, the FAD7 gene and the FAD8, or a nucleic acid sequence encoding it, and an editing protein or nucleic acid sequence encoding it; and a composition for controlling the content of a specific unsaturated fatty acid, which includes a complex formed by a guide nucleic acid capable of independently forming a complementary bond with the target sequence of one or more genes selected from the group consisting of a FAD2 gene, a FAD3 gene, a FAD6 gene, the FAD7 gene and the FAD8 gene, or the nucleic acid sequence encoding it and the editing protein.

Here, the guide nucleic acid or the nucleic acid sequence encoding it and the nucleic acid sequence encoding the editing protein may be present in the form of one or more vectors. They may be present in the form of a homologous or heterologous vector.

Method for controlling unsaturated fatty acid.

In another illustrative embodiment of the present invention, the present invention provides a method for controlling unsaturated fatty acid content, which comprises preparing the above-described composition and administering an effective amount of the composition to a target plant organism.

Gene manipulation.

A method of controlling the unsaturated fatty acid content by manipulating a gene of the organism may be used. Such a control method may involve introducing a composition intended for genetic manipulation into a plant organism for the purpose of manipulating a gene of the plant organism.

A composition intended for genetic manipulation may contain a guide nucleic acid-editing protein complex.

A composition intended for genetic manipulation may be introduced into a specific type of plant.

In this case, the particular type of plant may be a seed, but the present invention is not limited to it.

In one aspect, the present invention may relate to a method for modifying a target polynucleotide in plant cells.

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In one illustrative embodiment, the method involves obtaining cells or a population of cells from a plant as a sample and modifying the cells or population of cells. Cultivation can be performed at any stage outside the plant organism. The cell or cells can be reintroduced into the plant.

Additionally, in another exemplary embodiment, the present invention may relate to a method for artificially manipulating cells that includes:

introducing (a) a guide nucleic acid capable of forming a complementary bond with each of the target sequences of one or more genes selected from the group consisting of the FAD2 gene, the FAD3 gene, the FAD6 gene, the FAD7 gene and the FAD8 gene, or a nucleic acid sequence encoding the same; and (b) an editing protein comprising one or more proteins selected from the group consisting of a Cas9 protein derived from Streptococcus pyogenes, a Cas9 protein derived from Campylobacter jejuni, a Cas9 protein derived from Streptococcus thermophilus, a Cas9 protein derived from Staphylococcus aureus , the Neisseria meningitidis-derived Cas9 protein, and the Cpf1 protein, or a nucleic acid sequence encoding it, into plant cells.

The guide nucleic acid and editing protein may be present in one or more vectors in the form of a nucleic acid sequence or as a complex of a combination of guide nucleic acid and editing protein.

The methodology of Section 7. Delivery described above can be addressed before the administration stage.

For example, the introduction step can be carried out using one or more methods selected from a gene gun, electroporation, liposomes, plasmids, viral vectors, nanoparticles and a protein translocation domain (PTD) fusion method.

For example, the viral vector may be one or more selected from the group consisting of mosaic virus, retrovirus, lentivirus, adenovirus, adeno-associated virus (AAV), vaccinia virus, poxvirus, and herpes simplex virus.

For example, the vector can be incorporated into Agrobacterium and then introduced.

If an unsaturated fatty acid biosynthesis-associated factor is artificially manipulated using methods and compositions in accordance with certain exemplary embodiments of the present invention, the type and/or content of the unsaturated fatty acid can be controlled, e.g., increasing or decreasing the content of a specific unsaturated fatty acid and/or change the content of a specific unsaturated fatty acid, which means a plant can be obtained in which the content of an unsaturated fatty acid beneficial to human health increases, or the content of harmful unsaturated fatty acid decreases, and/or its processed product (food product, etc.) .d.).

Additional application options.

In any illustrative embodiment of the present invention, a kit for preparing a composition for controlling unsaturated fatty acid content may be provided, which includes the composition.

The kit can be prepared using a conventional preparation method known in the art.

The set may further include a detectable label. The term detectable label refers to an atom or molecule designed to specifically detect a molecule containing a label among the same type of molecules without a label. The detectable label may be attached to an antibody that specifically binds to the protein or fragment thereof, an interaction protein, a ligand, a nanoparticle, or an aptamer. The detectable label may include a radionuclide, a fluorophore, and an enzyme.

In any exemplary embodiment of the present invention, a method for screening material capable of controlling the expression level of one or more genes selected from the FAD2 gene, the FAD3 gene, the FAD6 gene, the FAD7 gene and the FAD8 gene, which are subject to artificial manipulation, may be provided.

In any illustrative embodiment of the present invention, a method of providing sequence information at a manipulated target location in a subject may be provided by analyzing the sequence of one or more genes selected from the group consisting of a FAD2 gene, a FAD3 gene, a FAD6 gene, a FAD7 gene, and FAD8 gene.

In addition, the present invention provides a method for constructing a library using the provided information.

In this case, a well-known database can be used.

In specific illustrative embodiments, the implementation of the present invention may provide a plant or cells that can be used for research using the method of the present invention.

Plant or cells that include edited chromosomes in one or more nucleic acid sequences associated with unsaturated fatty acid biosynthesis can be produced using the method of the present invention. Nuk sequence

- 74 046169 leic acid may be a sequence that may encode a protein sequence associated with the biosynthesis of an unsaturated fatty acid, or a reference sequence associated with the biosynthesis of an unsaturated fatty acid.

In one illustrative embodiment, the effect of the mutation and the mechanism of unsaturated fatty acid biosynthesis can be studied in a plant or cells using a measurement commonly used in a study related to unsaturated fatty acid biosynthesis using a plant or cells obtained using the method of the present invention. Alternatively, the effect of the active compound on unsaturated fatty acid biosynthesis can be studied using a plant or cells.

In another illustrative embodiment, the effect of an available gene manipulation strategy can be assessed using a plant or cells obtained using the method of the present invention. In other words, by modifying the chromosomal sequence encoding a protein associated with the biosynthesis of an unsaturated fatty acid, the biosynthesis of the corresponding unsaturated fatty acid can be stimulated or inhibited. In particular, this method involves the creation of a modified protein by editing the chromosomal sequence encoding a protein associated with the biosynthesis of an unsaturated fatty acid, resulting in modification of the plant or cells. Therefore, in some exemplary embodiments, the effect of using a genetically modified plant can be assessed by comparing the mechanism of unsaturated fatty acid biosynthesis with that of the wild type.

A genetically modified plant can be used to produce a plant organism with increased or decreased levels of specific unsaturated fatty acids through the use of an unsaturated fatty acid biosynthesis-associated factor and a system designed to control the unsaturated fatty acid whose function is thus artificially modified. By controlling the various mechanisms involved in a number of factors associated with unsaturated fatty acid biosynthesis, the system designed to control unsaturated fatty acid can be improved.

Examples

Hereinafter, the present invention will be described in further detail with reference to examples.

These examples are presented solely to describe the present invention in further detail, and those skilled in the art will appreciate that the scope of the present invention is not limited to the following examples.

Experimental methods.

1. Construction of gRNA.

The CRISPR/Cas9 target site of the soybean FAD2 gene was selected using CRISPR RGEN tools (Research Institute of Natural Sciences, South Korea). The target site of each gene may differ according to the type of CRISPR enzyme, and the target gene sequence for SpCas9 is briefly described in the above table. 1.

2. Vector design for soybean transformation.

In the soybean transformation test, the pPZP vector incorporating gRNA from FAD2-7 or FAD2-30 was used to target the FAD2 gene, and the vector also included Cas9. Agrobacterium tumefaciens strain EHA105 was transformed with the constructed vectors pPZP-FAD2-7 and pPZP-FAD2-30 (Fig. 1).

3. Soybean transformants and obtaining _T1 seeds.

1) Sterilization and soaking of seeds.

Seeds were sterilized using hydrochloric acid gas, prepared by mixing chlorine bleach (100 ml 12% sodium hypochlorite) with concentrated hydrochloric acid (12 N HCl, 5 ml) for 20 h, incubated in a 1% sodium hypochlorite suspension for 10 min to additional sterilization and washed with sterile water three times at intervals of 10 minutes. Each of the sterile seeds was placed in a 50 ml conical tube, and then sterile water was poured into the conical tube to soak at room temperature for 20 hours.

2) Teaching inoculum (Agrobacterium tumefaciens).

The soybean transformation test used vectors pPZP-FAD2-7 and pPZP-FAD2-30 constructed in Agrobacterium tumefaciens strain EHA105 and including PPTR. The bacterial strain containing the vector was streaked onto solid YEP medium [75 mg/L streptomycin, 25 mg/L rifampicin, 10 g/L peptone, 5 g/L NaCl, 5 g/L yeast extract, 1.5 wt. vol.% agar (pH 7.0)] and cultured at 28°C, thereby obtaining a separate colony. The colony was suspended in 10 ml of liquid YEP medium containing the same antibiotics contained in solid YEP medium and shaken at 220 rpm at 28°C until an OD ₆₅₀ of 0.6-0.8 was achieved. 10 ml of 30% glycerol stock solution was added and mixed with fully grown bacterial cells, 1 ml of cell suspension was added to each 1.5 ml tube, quickly cooled with liquid nitrogen and stored at -70°C. The day before inoculation was added

- 75 046 169 ml of Agrobacterium tumefaciens stock culture, which was stored at -70°C, in 200 ml of liquid YEP medium containing antibiotic, and cultured with shaking in a thermostat at 250 rpm at 25°C until an OD of ₆ 5 ₀ 0 was reached, 6-0.8. One day after inoculation, 200 ml of liquid YEP medium was divided into 50 ml and centrifuged at 20°C and 3270 g for 10 min. Added 15 ml of liquid co-culture medium (CCM; 0.32 g/L salt B5, 1.67 mg/L BA, 20 mM MES, 0.25 mg/L GA ₃ , 0.2 mM acetosyringone, 3.3 mM L-cysteine, 1.0 mM sodium thiosulfate, 1.0 mM DTT, 3% sucrose, pH 5.4) to the Agrobacterium tumefaciens cell pellet in each tube.

3) Inoculation and co-culture.

The soaked seed was then vertically cut down to the hypocotyl with a scalpel inserted between the two cotyledons and the seed coat was removed. The embryonic axis was cut approximately 1 cm below the cotyledon and one side to which the embryonic axis was attached was incised 7-8 times with a scalpel (no. 11 blade). In this case, the scalpel was coated with 15 ml of concentrate and the wound was applied to the target area. Approximately 50 explants were placed in 15 ml coculture with Agrobacterium tumefaciens and sonicated for 20 s and then inoculated for 30 min. After each explant was removed from the tube and placed on filter paper to remove moisture, a sheet of filter paper was placed on solid CCM (same as liquid CCM, agar (0.7%)) and then 10 explants were placed on it (placed adaxial side down ). The plate was covered with a microporous tape and co-cultured photoperiodically for 18 h at 25°C for 5 days.

4) Washing and inducing shoot formation.

Five days after co-culture, the explants were briefly washed with liquid germination induction medium 1/2 (SIM) for 10 min for sterilization. The explants were placed on filter paper to remove moisture, and then six explants per plate were placed in plates containing selective SI-® without antibiotics (germination inducing medium; 3.2 g/l salt B5, 1.67 mg/l BA, 3 mM MES, 0.8% agar, 3% sucrose, 250 mg/L cefotaxime, 50 mg/L vancomycin, 100 mg/L ticarcillin, pH 5.6), and the regenerating portion of the explant was positioned side up at an angle of approximately 30°. Each plate was covered with a microporous tape and cultured photoperiodically at 25°C for 18 h.

After two weeks, the germinated explants were placed on 10 mg/l selective antibiotic-containing PPT SI-® (same as S1-®, with the addition of 10 mg/l DL-phosphinothricin, pH 5.6), and the other part, except the seedling , was removed and placed so that the adaxial part was facing down.

5) Elongation of the shoot.

After two weeks, the germination substrate containing brown seedlings was cut with a scalpel (no. 15 blade) and placed on 5 mg/L selective antibiotic-containing PPT shoot elongation medium (SEM; 4.4 g/L MS salt, 3 mM MES, 0.5 mg/l GA ₃ , 50 mg/l asparagine, 100 mg/l pyroglutamic acid, 0.1 mg/l IAA, 1 mg/l zeatin, 3% sucrose, 0.6% agar, 250 mg/l cefotaxime, 50 mg/l vancomycin, 100 mg/l ticarcillin, 5 mg/l DL-phosphinothricin, pH 5.6). Every two weeks, the germination substrate with shoots was transferred to a fresh SEM, the brown part of the shoot was removed using the top of a scalpel (no. 15 blade) and the germination substrate was gradually scraped out to allow the medium to be well absorbed. When the shoot grew to the top of the Petri dish, two Petri dishes (100 mm x 40 mm) were stacked so that the shoot grew to approximately 8 cm. Each dish was covered with microporous tape and incubated photoperiodically at 25°C for 18 hours.

6) Rooting, acclimatization and coloring of leaves using PPT.

When the shoot length at SEM selection was 4 cm or greater, the seedling was excised with a scalpel (no. 11 blade) and transferred to rooting medium (RM; 4.4 g/L MS salt, 3 mM MES, 3% 0.8% sucrose). agar, 50 mg/l cefotaxime, 50 mg/l vancomycin, 50 mg/l ticarcillin, 25 mg/l asparagine, 25 mg/l pyroglutamic acid, pH 5.6). Here, the lower part of the elongated shoot, separated by cutting, was immersed in 1 mg/ml IBA for three minutes and then placed in a test tube containing RM.

When the root had grown sufficiently, the medium was washed out of the root with triple distilled water. The grown root was transplanted into a small pot (6 cm x 6 cm x 5.6 cm) containing a mixture of soil (Bio Plug No. 2, Heungnong seeding) and vermiculite (2:1), and placed in a Magenda box. After approximately 10 days, a solution of 100 mg/L DL-phosphinothricin was applied to the leaf surface.

7) Obtaining T1 seeds.

When the plant organism had grown sufficiently, it was transplanted into a larger pot and covered with a transparent plastic cap with approximately 10 pores. After 10 days, the plant organism was treated with Basta® (BAYER, 53 mg/l). As a result, non-transformant organisms (Glycine max L. Kwangan, NT) reacted sensitively and thus became weaker, while transformants did not show any changes and showed resistance. Nine soybean transformants (eight pPZP-FAD2-7 and one pPZP-FAD2-30) demonstrating resistance,

- 76 046169 was transferred to the greenhouse, resulting in T ₁ seeds.

8) Removing the gene introduced into the T1 transformant and obtaining T2 seeds.

To confirm that the gene introduced into the transgenic plant organism T1 was deleted, T1 seeds were sown and the plant organism was grown to 15 cm or more with 9 or more leaves in a small pot, followed by phosphinothricin staining of the leaves. The selected transgenic plant organism was transferred to a larger pot (20 cm (diameter) x 25 cm (height)) and grown in a greenhouse to obtain sample T1 and produce _T2 seeds.

4. Gene transfer analysis.

1 g of leaves of temporary transformants was weighed, frozen in liquid nitrogen, and the frozen leaves were finely ground in a mortar, and then genomic DNA was extracted using the CTAB method. To determine that the gene had been introduced, PCR was performed using the FAD2-7 and FAD2-30 gRNA sequences, the Bar selectable gene, and the Cas9 gene. The following primers were used to confirm the introduction of the FAD2-7 and FAD2-30 genes.

In the case of the FAD2-7 gene, a forward primer for AtU6p (5'-GAATGATTAGGCATCGAACC-3' (SEQ ID NO: 31) _and a reverse primer were used

FAD2-7 (5'-AAACTCCTCAAGGGTTCCAAACAC-3' (SEQ GO NO: 31)).

In the case of the FAD2-30 gene, the forward promoter primer (5'-GAATGATTAGGCATCGAACC-3' (SEQ ID NO: 31)) for the AtU6p promoter and the reverse primer (5'-AAACTCCTCAAGGGTTCCAAACACC-3 (SEQ ID NO: 33)) FAD2- 7. To confirm the introduction of the Bar gene, the Bar forward primer (5'-TCCGTACCGAGCCGCAGGAA-3' (SEQ ID NO: 34)) and the Bar reverse primer (5'-CCGGCAGGCTGAAGTCCAGC-3' (SEQ ID NO: 35)) were used.

In addition, to confirm the introduction of Cas9, PCR was performed using the following three sets of primers:

set with forward primer Cas9-® (5'-ATGGACAAGAAGTACAGCATCGGC-3' (SEQ ID NO: 36)) and reverse primer Cas9-® (5'-AACTTGTAGAACTCCTCCTGGCTG-3' (SEQ ID NO: 37));

set with direct primer

Cas9-@ (5'-TTCAGGAAGTCCAGGATGGTCTTG-3' (SEQ ID NO: 38" _and reverse primer Cas9-@ (5-AGAACTGGAAGTCCTTGCGGAAGT-3' (SEQ ID NO: 39));

as well as a set with direct primer

Cas9-@ (5'-CTGAGCGAGCTGGACAAGGCCGG-3'(SEQIDNO:40)) _and reverse primer Cas9-@ (5'-TTAGGCGTAGTCGGGCACGTCGTA-3' (SEQ ID NO: 41)).

5. Gene deletion analysis.

1 g of leaves of the transgenic plant organism T ₁ were weighed and frozen in liquid nitrogen, and the frozen leaves were finely ground in a mortar. Place 0.5 g of crushed leaves in a 2 ml test tube, add 1 ml of a solution obtained by mixing cetyltrimethylammonium buffer (cTAB) and β-mercaptoethanol (2-ME) in a ratio of 20:1 and process in a heating block at 65°C in for 1 hour, and then 10 μl of RNase (1.5 mg/150 μl H ₂ O) was added to ensure incubation at 37°C for 1 hour. After one hour, chloroform was mixed with isoamyl alcohol (24:1) in the same volume and mixed well to obtain the reagent, and then centrifuged at 4°C and 12000 rpm for 10 minutes. The supernatant was transferred to a new tube and again treated with the reagent chloroform:isoamyl alcohol (24:1). After adding the supernatant to the same volume of isopropanol and mixing by inverting 3-4 times, place it in a refrigerator at -20°C for 1 hour and centrifuge at 4°C and 12000 rpm for 10 minutes. After discarding the supernatant, 1 ml of 70% ethanol was added and the resulting solution was again centrifuged for 2 min to thereby obtain a cell pellet. After discarding the supernatant, the cell pellet was dried at room temperature for 20 min and then suspended in 30 μL of distilled water to extract genomic DNA. Extracted genomic DNA was diluted 20-fold and used as a template.

The base sequences of the introduced gene and the selected antibiotic resistance gene BAR were subjected to 35 PCR cycles, consisting of preliminary denaturation at 95°C for 10 min, denaturation at 95°C for 30 s, annealing at 55-65°C for 30 s and extension at 72°C for 30 s -1.5 min, were further extended at 72°C for 10 min, and the result was determined using a PCR kit (Prime Taq Premix, GENETBIO, Korea).

6. Analysis of oleic acid content.

For fatty acid analysis, three soybean seeds obtained from each transformant were analyzed separately. Each seed was placed in a paper bag, crushed using a hammer, and the fatty acid was extracted using 5 ml of extraction solvent (chloroform:hexane:methanol, 8:5:2) at room temperature for 12 hours. 150 μl of the extracted fatty acid was transferred to vial, 75 μl of methylation reagent (0.25 M methanolic sodium methoxide:petroleum ether:ethyl ether, 1:5:2) was added, and then hexane was added to 1 ml. Entered 1

- 77 046169 μl of sample and analyzed using a gas chromatography device (GC, Agilent Technologies, USA), and the analysis conditions are shown in table. 2 below. The fatty acid ratio was calculated by the area of each fatty acid in relation to the total area of fatty acids.

table 2

GC Conditions for Fatty Acid Analysis in Soybean

Paragraph Condition Tool Agilent 7890A Column 0.25μm(inner diameter)*30m capillary column DB-FFAP Detector Flame ionization detector Oven temperature 230°C Administration temperature 210°C Detector temperature 250°C Carrier gas N2 (1.5 ml/minute) Volume of administration 1 µl

7. Sequence analysis of the FAD2 gene.

PCR amplification of the target region of 200-300 bp was performed. using Hipi Plus DNA polymerase (Elpisbio). The PCR product obtained by the above method was sequenced using the MiSeq system (Illumina) and then analyzed using the Cas analyzer, CRISPR RGEN tool (www.rgenome.net).

Example 1. Vector for transformation and obtaining a transformant.

For knockout of the FAD2 gene, as shown in Table. 1, a guide RNA targeting the FAD2 gene was developed and cloned with the Cas9 protein into the pPZP vector, thereby constructing a soybean transformation vector (FIG. 1). As shown in FIG. 2, using the regeneration process, the plants obtained a total of 9 transformants (To) (eight pPZP-FAD2-7 8 and one pPZP-FAD230) (Fig. 3). T ₁ seeds were collected from T ₀ transformants, thereby obtaining T ₁ transformants (Fig. 10).

Example 2. PCR analysis to determine gene transfer into a transformant.

DNA was isolated from FAD2-7 and FAD2-30 T0 transformants and PCR was performed according to the method described above, confirming complete introduction of the introduced guide RNA, FAD2-7 and FAD2-30 and the selection gene Bar and Cas9 (Fig. 4).

Example 3. Analysis of oleic acid content in transgenic soybean.

Oleic acid contents were analyzed in FAD2-7 and FAD2-30 T1 seeds according to the above method. The oleic acid contents of FAD2-7 and FAD2-30 T ₁ seeds were significantly higher than those of wild-type seeds such as Glycine max L. Pungsan, Glycine max L. Kwangan and Glycine max L. Hosim (Fig. 5).

Example 4. Sequence analysis of the FAD2 gene in transgenic soybean.

The frequency of insertion-deletion mutations (Fig. 6) and sequences (Fig. 7) were analyzed to confirm the induction of mutation in the FAD2 genes FAD2-7 and FAD2-30 of T ₁ seeds according to the above method. Confirmed that a mutation was induced in FAD2-7 seed T ₁

In addition, T ₁ transformants were analyzed by deep sequencing to confirm the induction of a mutation in the FAD2 gene. As a result, it was confirmed that the mutation was induced in the target site of the FAD2 gene in chromosome No. 10 (chr10) and chromosome No. 20 (chr20) of other T1 transformants with the exception of FAD2-7 No. 1-1 and FAD2-30 No. 2-4, No. 9- 1 and No. 3-1 (Fig. 8 and 9).

Example 5. Analysis of the removal of the introduced transgenic soybean gene.

To confirm the removal of the gene from the selected T ₁ transformants, PCR was performed for the selectable BAR gene and the gene introduced as part of the input vector. As a result, it was confirmed that both introduced genes and the BAR gene were removed from one FAD2-7 T1 transformant, and partial removal was confirmed from fifteen FAD2-30 T1 transformants in individuals (9-1, 19-1, 21-1, 21-5) genes (Fig. 11).

Industrial applicability

The processed product can be manufactured using a plant organism with an increased content of a specific unsaturated fatty acid that is beneficial to human health, or with a reduced content of a specific unsaturated fatty acid that is harmful to human health, using an artificial unsaturated fatty acid biosynthesis-associated factor manipulation, and a system designed to control the unsaturated fatty acid, which is thus artificially modified and can be used for food.

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Claims (14)

CLAIM 1. A plant containing a manipulated genome, wherein the manipulated genome comprises a manipulated FAD2 gene in which an insertion-deletion mutation is induced in a sequence selected from SEQ ID NO: 1-30 by means of a CRISPR/Cas system, wherein the sequence, selected from SEQ ID NO: 1-30, contains the PAM sequence 5'NGG-3', where N is independently A, T, C or G, wherein the manipulated FAD2 gene is capable of knocking out the wild type FAD2 gene, thereby inhibiting expression a wild-type FAD2 gene in a plant, wherein the plant containing the artificially manipulated gene, compared to the wild-type plant, is characterized by at least one phenotype selected from the group consisting of: (a) decreased FAD2 protein expression; (b) reduced amount of RNA transcripts of the FAD2 gene; (c) increased content of C8-24:D1 unsaturated fatty acid; and (d) decreased content of C8-24:D2 unsaturated fatty acid. 2. The plant of claim 1, wherein the C8-24:D1 unsaturated fatty acid is a C16-22:D1 unsaturated fatty acid. 3. The plant of claim 1, wherein the C8-24:D1 unsaturated fatty acid is a C18:D1 unsaturated fatty acid. 4. The plant of claim 1, wherein the C8-24:D2 unsaturated fatty acid is a C16-22:D2 unsaturated fatty acid. 5. The plant of claim 1, wherein the C8-24:D2 unsaturated fatty acid is a C18:D2 unsaturated fatty acid. 6. The plant according to claim 1, wherein the plant is soybean. 7. A composition for producing a plant containing an artificially manipulated genome, which contains: a guide nucleic acid capable of targeting the FAD2 gene, or a nucleic acid sequence encoding the same, and an editing protein or a nucleic acid sequence encoding the same, where the editing protein is a Cas9 protein derived from Streptococcus pyogenes, where the guide nucleic acid is an RNA sequence capable of binding a target region within the nucleic acid sequence of the FAD2 gene, wherein the target gene region contains at least one nucleotide sequence selected from SEQ ID NO: 1-30. 8. The composition according to claim 7, wherein the composition for gene manipulation is formulated in an Agrobacterium-based vector system. 9. The composition according to claim 7, wherein the composition intended for gene manipulation is formulated in a viral vector system. 10. The composition of claim 9, wherein the viral vector includes one or more viral vectors selected from mosaic virus, retrovirus, lentivirus, adenovirus, adeno-associated virus (AAV), vaccinia virus, poxvirus and herpes simplex virus. 11. A method for producing a plant containing an artificially manipulated genome according to claim 1, comprising delivering the composition to a subject, where the subject is a plant cell, a seed, a part of a plant organism or an entire plant organism, where the composition contains: a guide nucleic acid capable of targeting the FAD2 gene, or a nucleic acid sequence encoding the same, and an editing protein or a nucleic acid sequence encoding the same, where the editing protein is a Cas9 protein derived from Streptococcus pyogenes, where the guide nucleic acid is an RNA sequence capable of binding a target region within the nucleic acid sequence of the FAD2 gene, wherein the target gene region contains at least one nucleotide sequence selected from SEQ ID NO: 1-30. 12. The method according to claim 11, where the composition is formulated in a vector system based on Agrobacterium. 13. The method according to claim 11, where the composition is composed in a viral vector system. 14. The method of claim 13, wherein the viral vector includes one or more viral vectors selected from mosaic virus, retrovirus, lentivirus, adenovirus, adeno-associated virus (AAV), vaccinia virus, poxvirus and herpes simplex virus. -